Self-regulated learning profiles including test anxiety linked to stress and performance: A latent profile analysis based across multiple cohorts.

INTRODUCTION: Medical educators aim to understand why students differ in performance and stress. While performance and stress are associated with student demographics, school factors and aspects of self-regulated learning (SRL), it remains unclear how these elements interact within individuals. This multi-cohort study identified SRL profiles among medical students and explored their associations with performance and stress. Additionally, we examined the identified profiles' associations with gender, migration status and assessment policy. METHODS: We used latent profile analysis (LPA) to identify profiles on Motivated Strategies for Learning Questionnaire (MSLQ) scores of six cohorts (2014-2019) of Year 1, first semester medical students (n = 1894) in a Dutch medical school. We used nine MSLQ subscales that measure test anxiety (TA), self-efficacy, deep learning, resource management and value. The university's assessment policy varied, demanding students to obtain 100% or 75% of Year 1 credits to remain enrolled. We defined optimal performance as obtaining all credits at the end of Year 1. Two cohorts completed the Perceived Stress Scale (PSS-14, n = 409) in the 2nd semester. RESULTS: We identified three distinct student profiles: 693/1894(36.6%) were classified as TAhighSRLhigh, 661/1894(34.9%) as TAlowSRLhigh and 540/1894(28.5%) as TAmoderateSRLlow. Females were more likely to belong to TAhighSRLhigh profiles compared to males (effect size [ES] Cramer's V = .13, small). Migration background was not associated with these profiles. The TAhighSRLhigh profile was more prevalent under the 100% assessment policy (ES Cramer's V = .10, negligible). TAlowSRLhigh students demonstrated lower stress levels (PSS = 23.9 out of 56) compared to TAhighSRLhigh students (PSS = 28.7, ES Cohen's d = .62, medium) and TAmoderateSRLlow students (PSS = 28.2, ES Cohen's d = .51, medium). Performance differed among the three profiles (ES Cramer's V = .16, small): 82.5% optimal performance in the TAlowSRLhigh, 71.9% in the TAhighSRLhigh and 65.2% in the TAmoderateSRLlow profile. DISCUSSION: Three distinct SRL student profiles associated with gender, academic performance and perceived stress were identified. Test anxiety had additional value in distinguishing subgroups with differential academic performance and stress. These profiles may aid educators to inform personalised support strategies for novice learners.

The association between how medical students were selected and their perceived stress levels in Year-1 of medical school.

BACKGROUND: The prevalence of medical students' mental distress is high. While schools apply various methods to select a well-performing and diverse student population, little is known about the association between different selection methods and the well-being of these students during medical school. The present retrospective multi-cohort study assessed whether students selected by high grades, assessment, or weighted lottery showed different stress perception levels in Year-1 of medical school. METHODS: Of 1144 Dutch Year-1 medical students, 650 (57%) of the cohorts 2013, 2014, and 2018 who were selected by high grades, assessment, or weighted lottery completed a stress perception questionnaire (PSS-14). A multilevel regression analysis assessed the association between selection method (independent variable) and stress perception levels (dependent variable) while controlling for gender and cohort. In a post-hoc analysis, academic performance (optimal vs. non-optimal) was included in the multilevel model. RESULTS: Students selected by assessment (B = 2.25, p < .01, effect size (ES) = small) or weighted lottery (B = 3.95, p < .01, ES = medium) had higher stress perception levels than students selected by high grades. Extending the regression model with optimal academic performance (B=-4.38, p < .001, ES = medium), eliminated the statistically significant difference in stress perception between assessment and high grades and reduced the difference between weighted lottery and high grades from 3.95 to 2.45 (B = 2.45, p < .05, ES = small). CONCLUSIONS: Selection methods intended to create a diverse student population - assessment and lottery - are associated with higher stress perception levels in Year-1 of medical school. These findings offer medical schools insights into fulfilling their responsibility to take care of their students' well-being.

Medical students' crisis-induced stress and the association with social support.

BACKGROUND: Medical schools are challenged to guard student wellbeing given the potential negative impact of the COVID-19 outbreak combined with an already high prevalence of mental distress. Although social support is generally associated with less crisis-induced stress, it is unknown whether this applies to medical students during the COVID-19 outbreak. OBJECTIVES: The impact of the COVID-19 outbreak on perceived stress of medical students was assessed by comparing their perceived stress levels during the outbreak to both their own baseline and the previous cohort's pre-COVID-19 stress levels. Then, the association between social support and stress during the COVID-19 outbreak was assessed. METHODS: Dutch Year-1 medical students of cohort 2019 (n = 99) completed the 14-item Perceived Stress Scale (PSS-14) at two time points: baseline (pre-COVID-19) and final measurement (COVID-19). Social support-emotional-informational support and club membership-was assessed during the final measurement. PSS and social support scores were compared to similar measurements of cohort 2018 (n = 196). Students' baseline stress levels, gender, and study performance were controlled for when comparing final stress levels. RESULTS: In cohort 2018 (pre-COVID-19), students' perceived stress levels did not differ significantly between the baseline and final measurements. Additionally, baseline stress levels of the two cohorts (2018 and 2019) were not found to be significantly different. Cohort 2019's final stress levels (COVID-19) were significantly higher compared to their baseline stress levels (paired t-test: t = 6.07, p < .001) and cohort 2018's final stress levels (linear regression: B = 4.186, p < .001). Only during the COVID-19 outbreak higher social support levels-i.e., emotional-informational support (B = -0.75, p < .001) and club membership (B = -3.68, p < .01)-were associated with lower stress levels. CONCLUSIONS: During the COVID-19 outbreak, medical students' perceived stress levels were higher-especially for students with lower social support levels. Our results suggest that medical schools should optimize social support to minimize crisis-induced stress.

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.

Effects of raising the bar on medical student study progress: An intersectional approach.

CONTEXT: Medical schools seek for measures to improve their students' study progress and are responsible for a diverse student population. OBJECTIVES: The effect of a stricter academic dismissal (AD) policy in medical school on short-term and long-term study progress was investigated in a longitudinal cohort study. In addition, differential effects for subgroups were assessed by intersecting gender, ethnicity and prior education (intersectional framework). METHODS: Participants were first-year Bachelor students enrolled in 2011 to 2016 in a Dutch medical school. For cohorts 2011-2013, the AD policy consisted of a minimum of 67% of Year-1 credits required to remain enrolled (67%-policy, n = 1189), and for cohorts 2014-2016, this bar was raised to 100% of Year-1 credits (100%-policy, n = 1233). Outcome measures on study progress were Year-1 completion and dropout (short term) and Bachelor completion in three and four years (long term). RESULTS: Overall, Year-1 completion rates increased under the 100%-policy compared to the 67%-policy (OR = 2.50, 95%-CI:2.06-3.03, P < .001). Yet, this increase was not present for students with non-standard prior education - except for males with a migration background (OR = 7.19, 95%-CI:2.33-25.73, P < .01). The dropout rate doubled under the 100%-policy (OR = 2.41, 95%-CI:1.68-3.53, P < .001). Mainly students with standard prior education dropped out more often (OR = 3.68, 95%-CI:2.37-5.89, P < .001), except for males with a migration background. Bachelor completion rates after three and four years were not positively affected by the 100%-policy. Notably, females without a migration background and with non-standard prior education suffered from the 100%-policy regarding Bachelor completion after three years (OR = 0.29, 95%-CI:0.11-0.76, P < .05). CONCLUSIONS: Despite increased dropout rates, the stricter AD policy improved Year-1 completion rates - especially for under-represented subgroups, thereby improving study progress without harming student diversity on the short term. However, these positive effects did not hold regarding Bachelor completion rates indicating that long-term effects require higher performance standards throughout the Bachelor, which in turn may harm other subgroups and thereby student diversity.

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.

Comparison of the PU.1 transcriptional regulome and interactome in human and mouse inflammatory dendritic cells.

Dendritic cells (DCs) are key immune modulators and are able to mount immune responses or tolerance. DC differentiation and activation imply a plethora of molecular and cellular responses, including transcriptional changes. PU.1 is a highly expressed transcription factor in DCs and coordinates relevant aspects of DC biology. Due to their role as immune regulators, DCs pose as a promising immunotherapy tool. However, some of their functional features, such as survival, activation, or migration, are compromised due to the limitations to simulate in vitro the physiologic DC differentiation process. A better knowledge of transcriptional programs would allow the identification of potential targets for manipulation with the aim of obtaining "qualified" DCs for immunotherapy purposes. Most of the current knowledge regarding DC biology derives from studies using mouse models, which not always find a parallel in human. In the present study, we dissect the PU.1 transcriptional regulome and interactome in mouse and human DCs, in the steady state or LPS activated. The PU.1 transcriptional regulome was identified by performing PU.1 chromatin immunoprecipitation followed by high-throughput sequencing and pairing these data with RNAsequencing data. The PU.1 interactome was identified by performing PU.1 immunoprecipitation followed by mass spectrometry analysis. Our results portray PU.1 as a pivotal factor that plays an important role in the regulation of genes required for proper DC activation and function, and assures the repression of nonlineage genes. The interspecies differences between human and mouse DCs are surprisingly substantial, highlighting the need to study the biology of human DCs.

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.

Gender-specific effects of raising Year-1 standards on medical students' academic performance and stress levels.

CONTEXT: Medical schools are challenged to create academic environments that stimulate students to improve their study progress without compromising their well-being. OBJECTIVES: This prospective comparative cohort study investigated the effects of raising Year-1 standards on academic performance and on students' chronic psychological and biological stress levels. METHODS: In a Dutch medical school, students within the last Bachelor's degree cohort (n = 410) exposed to the 40/60 (67%) credit Year-1 standard (67%-credit cohort) were compared with students within the first cohort (n = 413) exposed to a 60/60 (100%) credit standard (100%-credit cohort). Main outcome measures were Year-1 pass rate (academic performance), mean score on the Perceived Stress Scale (PSS, psychological stress) and hair cortisol concentration (HCC, biological stress). RESULTS: Year-1 pass rates were significantly higher in the 100%-credit cohort (odds ratio [OR] 4.65). Interestingly, there was a significant interaction effect (OR 0.46), indicating that raising the standard was more effective for male than for female students. PSS scores (n = 234 [response rate [RR]: 57%] and n = 244 [RR: 59%] in the 67%- and 100%-credit cohorts, respectively) were also significantly higher in the 100%-credit cohort (F(1,474)  = 15.08, P < .001). This applied specifically to female students in the 100%-credit cohort. Levels of HCC (n = 181 [RR: 44%] and n = 162 [RR: 39%] respectively) did not differ between cohorts, but were significantly higher in female students (F(1,332)  = 7.93, P < .01). In separate models including cohort and gender, both PSS score (OR 0.91) and HCC (OR 0.38) were significantly associated with Year-1 performance. Only students with both high PSS scores and high HCC values were significantly at risk of lower Year-1 pass rates (OR 0.27), particularly male students. CONCLUSIONS: Raising the Year-1 performance standard increased academic performance, most notably in male students. However, it also increased levels of perceived stress, especially in female students. In particular, the combination of high levels of perceived stress and biological stress, as measured by long-term cortisol, was related to poor academic performance. The study suggests a relationship between raising performance standards and student well-being, with differential effects in male and female students.

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.

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.

Transcriptional patterns associated with BDCA3 expression on BDCA1+ myeloid dendritic cells.

Myeloid dendritic cells, including BDCA3hi DCs and BDCA1+ DCs (hereafter dubbed DC1 and DC2 for clarity), play a pivotal role in the induction and regulation of immune responses. Interestingly, a fraction of DC2 also express low to intermediate levels of BDCA3. It is unknown whether BDCA3+ DC2 also share other traits with DC1 that are absent in BDCA3- DC2 and/or whether BDCA3 expression renders DC2 functionally distinct from their BDCA3-lacking counterparts. Here, we used expression analysis on a predefined set of immunology-related genes to determine divergence between BDCA3-positive and BDCA3-negative DC2 and their relation to bona fide BDCA3hi DC1. Results showed that mRNA fingerprints of BDCA3+ DC2 and BDCA3- DC2 are very similar, and clearly distinct from that of DC1. Differences in mRNA expression, however, were observed between BDCA3+ DC2 and BDCA3- DC2 that pointed toward a more activated status of BDCA3+ DC2. In line with this, higher steady state maturation marker expression and TLR-induced maturation marker expression and inflammatory cytokine production by BDCA3+ DC2 were observed. This dataset provides insight into the relationship between myeloid DC populations and contributes to further understanding of DC immunobiology.

HBV-Derived Synthetic Long Peptide Can Boost CD4+ and CD8+ T-Cell Responses in Chronic HBV Patients Ex Vivo.

Background: Vaccination with synthetic long peptides (SLP) is a promising new treatment strategy for chronic hepatitis B virus (CHB). SLP can induce broad T-cell responses for all HLA types. Here we investigated the ability of a prototype HBV-core (HBc)-sequence-derived SLP to boost HBV-specific T cells in CHB patients ex vivo. Methods: HBc-SLP was used to assess cross-presentation by monocyte-derived dendritic cells (moDC) and BDCA1+ blood myeloid DC (mDC) to engineered HBV-specific CD8+ T cells. Autologous SLP-loaded and toll-like receptor (TLR)-stimulated DC were used to activate patient HBc-specific CD8+ and CD4+ T cells. Results: HBV-SLP was cross-presented by moDC, which was further enhanced by adjuvants. Patient-derived SLP-loaded moDC significantly increased autologous HBcAg18-27-specific CD8+ T cells and CD4+ T cells ex vivo. HBV-specific T cells were functional as they synthesized tumor necrosis factor-alpha and interferon-gamma. In 6/7 of patients blockade of PD-L1 further increased SLP effects. Also, importantly, patient-derived BDCA1+ mDC cross-presented and activated autologous T-cell responses ex vivo. Conclusions: As a proof of concept, we showed a prototype HBc-SLP can boost T-cell responses in patients ex vivo. These results pave the way for the development of a therapeutic SLP-based vaccine to induce effective HBV-specific adaptive immune responses in CHB patients.

The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function.

Chronic hepatitis B virus (HBV) infection results from inadequate HBV-specific immunity. BDCA3+ dendritic cells (DCs) are professional antigen presenting cells considered to be important for antiviral responses because of specific characteristics, including high interferon-λ production. BDCA3+ DCs may thus also have a role in the immune response against HBV, and immunotherapeutic strategies aiming to activate DCs, including BDCA3+ DCs, in patient livers may represent an interesting treatment option for chronic HBV. However, neither the effect of chronic hepatitis B (CHB) infection on the frequency and function of BDCA3+ DCs in liver and blood, nor the effect of the viral surface protein (HBsAg) that is abundantly present in blood of infected individuals are known. Here, we provide an overview of BDCA3+ DC frequency and functional capacity in CHB patients. We find that intrahepatic BDCA3+ DC numbers are increased in CHB patients. BDCA3+ DCs from patient blood are not more mature at steady state, but display an impaired capacity to mature and to produce interferon-λ upon polyI:C stimulation. Furthermore, in vitro experiments exposing blood and intrahepatic BDCA3+ DCs to the viral envelope protein HBsAg demonstrate that HBsAg does not directly induce phenotypical maturation of BDCA3+ DCs, but may reduce IFN-λ production via an indirect unknown mechanism. These results suggest that BDCA3+ DCs are available in the blood and on site in HBV infected livers, but measures may need to be taken to revive their function for DC-targeted therapy.

Similar frequencies, phenotype and activation status of intrahepatic NK cells in chronic HBV patients after long-term treatment with tenofovir disoproxil fumarate (TDF).

BACKGROUND: Currently, much effort is directed at further improving treatment for chronic hepatitis B patients by assessing the effect of immunomodulatory agents during therapy with nucleotide analogues (NUC). Although there are some reports on the effect of NUC therapy on peripheral natural killer (NK) cells, no studies investigated the long-term effects of NUC treatment on intrahepatic NK cells of chronic HBV patients. We aimed to prospectively investigate cell frequencies, phenotype, and activation status of intrahepatic NK cells of CHB patients on prolonged treatment with TDF. METHODS: Fine needle aspiration biopsies were collected from 11 chronic HBV patients at baseline, and at 12, 24, and 48 weeks of treatment with a daily 245 mg dose of TDF. Four patients underwent an additional aspiration biopsy after appoximately 6 years of treatment. RESULTS: Longitudinal evaluation of these patients during tenofovir therapy showed that all patients achieved a viral load reduction with undetectable DNA load after 48 weeks of therapy. Repeated sampling of the liver during therapy showed that the frequency of distinct lymphocyte populations in the liver remained unchanged despite viral load reduction. During the course of therapy, no modulation of the expression levels and frequencies of CD69, HLA-DR, NKG2A and NKG2D on liver NK cells were detected. However, evaluation of intrahepatic NK cell activation after continuous TDF therapy for 6 years demonstrated a mild increase in 3 out of 4 patients. CONCLUSIONS: Our findings provide a unique insight in the intrahepatic NK cell compartment in chronic HBV patients during prolonged treatment. We observed that long-term NUC-induced viral suppression, accompanied by gradual decrease of HBsAg levels, had no or only a limited effect on the frequencies, phenotype, and activation status of intrahepatic NK cells.

Hepatitis B Virus Surface Antigen Activates Myeloid Dendritic Cells via a Soluble CD14-Dependent Mechanism.

UNLABELLED: Hepatitis B virus (HBV) infection can cause chronic liver disease, which is associated with increased risk of liver cirrhosis, liver failure, and liver cancer. Clearance of HBV infection requires effective HBV-specific immunity; however, the immunological mechanisms that determine the development of effective HBV-specific immunity are poorly understood. Dendritic cells (DC) play a pivotal role in the regulation of antiviral immunity. Here, we investigated the interaction between HBV surface antigen (HBsAg), the main envelope glycoprotein of HBV, and BDCA1(+) myeloid dendritic cells (mDC). Exposure of peripheral blood-derived BDCA1(+) mDC to HBsAg resulted in strong DC maturation, cytokine production, and enhanced capacity to activate antigen-specific cytotoxic T cells (CTLs). By using neutralizing antibodies, crucial roles for CD14 and Toll-like receptor 4 (TLR4) in HBsAg-mediated BDCA1(+) mDC maturation were identified. Concordantly, HBsAg-mediated DC maturation required fetal calf serum (FCS) or human plasma, naturally containing soluble CD14 (sCD14). Intriguingly, HBsAg-induced DC maturation was significantly reduced in umbilical cord blood plasma, which contained less sCD14 than adult plasma, indicating that sCD14 is an important host factor for recognition of HBsAg by DC and subsequent DC activation. A direct interaction between sCD14 and HBsAg was demonstrated by using enzyme-linked immunosorbent assay (ELISA). Moreover, sCD14-HBsAg complexes were detected both in vitro and in sera of HBV-infected patients. The abundance of sCD14-HBsAg complexes varied between chronic HBV disease stages and correlated with activation of BDCA1(+) mDC in vivo We conclude that HBsAg activates BDCA1(+) DC via an sCD14-dependent mechanism. These findings provide important novel insights into the initiation of HBV-specific immunity and facilitate development of effective immunotherapeutic interventions for HBV. IMPORTANCE: Hepatitis B virus (HBV) infection is a significant health problem, as it causes progressive liver injury and liver cancer in patients with chronic HBV infection, which affects approximately 250 million individuals worldwide. Some of the infected adults and the majority of neonates fail to mount an effective immune response and consequently develop chronic infection. The viral and host factors involved in the initiation of effective HBV-specific immune responses remain poorly understood. Here we identified CD14 and TLR4 as receptors for HBsAg, the main HBV envelope antigen. HBsAg induced strong maturation of dendritic cells (DC), which have a central role in regulation of virus-specific immunity. These results provide essential novel insights into the mechanisms underlying the initiation of HBV-specific immunity. Intriguingly, since neonates have naturally low sCD14, the finding that serum-derived sCD14 is a crucial host factor for recognition of HBsAg by DC may have implications for immunity of neonates to HBV infection.

BDCA3 expression is associated with high IFN-λ production by CD34(+)-derived dendritic cells generated in the presence of GM-CSF, IL-4, and/or TGF-ß.

High BDCA3 expression is associated with a specific human IFN-λ-producing dendritic cell (DC) subset. However, BDCA3 has also been detected on other DC subsets. Thus far, development and function of BDCA3 expression on DCs remains poorly understood. Human Langerhans cells (LCs) and interstitial DCs (intDCs) can be generated in vitro by differentiation of CD34(+) hematopoietic progenitors via distinct precursor DCs (preDCs), CD1a(+) preDCs, and CD14(+) preDCs, respectively. Here, we identified BDCA3 expression in this well-known GM-CSF/TNF-α-driven culture system and described the effect of IL-4 and/or TGF-ß on induction of BDCA3 expression. In control or TGF-ß cultures, BDCA3 was only detected on CD14(+) preDC-derived intDCs. IL-4 induced BDCA3 expression in both CD14(+)-derived and CD1a(+)-derived cultures. TGF-ß and IL-4 together further increased CD14(+)-derived and CD1a(+)-derived BDCA3(+) DC frequencies, which partly expressed CLEC9A, but were not identical to the BDCA3(high) CLEC9A(+) DC subset in vivo. Importantly, BDCA3(+) cells, but not BDCA3(-) cells, in this system produced high IFN-λ levels upon polyinosinic:polycytidylic acid (polyI:C) stimulation. This culture system, in which BDCA3 expression is preferentially associated with the intDC lineage and IFN-λ-producing capacity, will greatly contribute to further research on the function and regulation of BDCA3 expression and IFN-λ production by DCs.