The effect of interferons and viral proteins on antigen-presenting cells in chronic hepatitis B

__Abstract__ The innate immune system forms the so-called first line of defense against invading pathogens like viruses. Innate immune cells include phagocytes like monocytes, macrophages and dendritic cells (DC). Phagocytes sample their environments, binding and taking up viral pathogens. By means of surface and intracellular recognition receptors these cells are able to recognize and react to viral pathogens by producing molecules like cytokines that attract and activate other immune cells. Furthermore, they can present pathogen-derived antigens to T cells. Therefore, they are also called antigen-presenting cells (APC). APC can induce T effector cell, but also B cell, activation and differentiation at the site of inflammation, ultimately leading to the induction of virus-specific T and B cell responses. Also in HBV infection, APC are key players in the initiation and regulation of antiviral immune responses, ensuing in combined cellular and humoral immunity that ultimately allows control of HBV infection. This thesis focuses on specific aspects of the role of APC in chronic HBV infection. Interferons, a family of cytokines, are used as antiviral therapy in chronic HBV infection, but what the exact effects of IFN on APC are and how this influences the role of APC in HBV infection remains elusive. Furthermore, APC are constantly exposed to viral proteins, but reports on their interaction with APC are scarce while literature on their effects on APC seem contradictive and/or insufficient. Therefore, we investigated effects of IFN and viral proteins on APC in chronic HBV infection to further define the role of APC in HBV infection

Monocytes from chronic HBV patients react in vitro to HBsAg and TLR by producing cytokines irrespective of stage of disease

Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation

Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program ‘iTHER’

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival

Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program ‘iTHER’

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival

IFN-λ-mediated IL-12 production in macrophages induces IFN-γ production in human NK cells

With increasing interest in alternative options to interferon-alpha-based treatments, IFN-λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN-λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN-λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN-λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon-alpha, IFN-λ is unable to directly stimulate NK cells, due to the absence of IFN-λ receptor chain 1 (IFN-λR1) on NK cells. However, IFN-λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL-12 family of cytokines in monocyte-derived macrophages. We further show that through macrophage-mediated IL-12 production, IFN-λ is able to indirectly affect NK cells and ultimately induce IFN-γ production

Monocytes from Chronic HBV Patients React <i>In Vitro</i> to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease

<div><p>Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the <i>in vitro</i> effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected <i>in vivo</i>. We observed that <i>in vitro</i> HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier <i>in vivo</i> exposure to HBsAg does not affect the <i>in vitro</i> response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production <i>in vitro</i>. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive <i>in vivo</i> monocyte activation.</p></div

Chronic HBV patients divided into three groups based on HBV DNA and HBeAg levels.

<p>HBeAg levels, HBsAg levels and HBV DNA were measured in 45 chronic HBV patients. (<b>A</b>) HBV patients were divided into an HBeAg^negative/HBeAg^low group (<100 IU/ml), and an HBeAg^high group (>100 IU/ml). Based on these two groups and HBV DNA levels, three groups of chronic HBV patients were defined (groups 1, 2 and 3). (<b>B</b>) Groups 1–3 were compared based on HBV DNA levels, HBeAg levels and HBsAg levels. *** p<0.0001, Kruskal-Wallis test, followed by Dunn's multiple comparison test.</p

Different HBV DNA, HBeAg and HBsAg levels do not alter the frequency of monocyte subpopulations.

<p>Whole blood samples from chronic HBV patients were stained for CD14 and CD16 to analyze frequencies of the monocyte CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ subpopulations. (<b>A</b>) Gating strategy: monocytes were identified on the basis of their forward-sideward scatter, and divided into CD14⁺⁺CD16^- and CD14⁺CD16⁺ populations. (<b>B</b>) The frequencies of monocyte subpopulations were compared between groups 1–3 (n = 45).</p

IL-10 inhibits the frequency of monocytes producing cytokines upon HBsAg exposure.

<p>PBMC from healthy individuals were stimulated with HBsAg, with or without IL-10. After overnight incubation, cells were stained for CD14 and intracellularly for cytokines. The frequencies of IL-6-positive (<b>A</b>) and TNF-positive (<b>B</b>) monocytes were compared between HBsAg and HBsAg and IL10 (n = 3).</p

HBsAg induces cytokine production by monocytes.

<p>PBMC from healthy individuals were stimulated with HBsAg. After overnight incubation, cells were stained for CD14, IL-6 and TNF. (<b>A</b>) Gating strategy: Viable monocytes were identified on the basis of their forward-sideward scatter profile and their membrane expression of CD14. (<b>B</b>) Representative intracellular cytokine stainings are presented showing IL-6 and TNF-producing monocytes upon incubation with medium and HBsAg. (<b>C</b>) The frequencies of monocytes producing IL-6 or TNF upon incubation with medium or HBsAg are presented (n = 9, ** p<0.01, Wilcoxon signed rank test).</p