The monocyte-derived cytokine response in whole blood from preterm newborns against sepsis-related bacteria is similar to term newborns and adults.

Introduction: Sepsis is characterized by a dysregulated innate immune response. It is a leading cause of morbidity and mortality in newborns, in particular for newborns that are born premature. Although previous literature indicate that the pro-inflammatory response may be impaired in preterm newborns, serum levels of monocyte-derived cytokines, such as TNF-α and IL-6, vary highly between newborns and can reach adult-like concentrations during sepsis. These contradictory observations and the severe consequences of neonatal sepsis in preterm newborns highlight the need for a better understanding of the pro-inflammatory cytokine response of preterm newborns to improve sepsis-related outcomes. Methods and results: Using an in vitro model with multiple read outs at the transcriptional and protein level, we consistently showed that the monocyte-derived cytokine response induced by sepsis-related bacteria is comparable between preterm newborns, term newborns and adults. We substantiated these findings by employing recombinant Toll-like receptor (TLR) ligands and showed that the activation of specific immune pathways, including the expression of TLRs, is also similar between preterm newborns, term newborns and adults. Importantly, we showed that at birth the production of TNF-α and IL-6 is highly variable between individuals and independent of gestational age. Discussion: These findings indicate that preterm newborns are equally capable of mounting a pro-inflammatory response against a broad range of bacterial pathogens that is comparable to term newborns and adults. Our results provide a better understanding of the pro-inflammatory response by preterm newborns and could guide the development of interventions that specifically modulate the pro-inflammatory response during sepsis in preterm newborns.

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.

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.