NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines.

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

Immunotherapeutic Potential of TGF-ß Inhibition and Oncolytic Viruses.

In cancer immunotherapy, a patient's own immune system is harnessed against cancer. Immune checkpoint inhibitors release the brakes on tumor-reactive T cells and, therefore, are particularly effective in treating certain immune-infiltrated solid tumors. By contrast, solid tumors with immune-silent profiles show limited efficacy of checkpoint blockers due to several barriers. Recent discoveries highlight transforming growth factor-ß (TGF-ß)-induced immune exclusion and a lack of immunogenicity as examples of these barriers. In this review, we summarize preclinical and clinical evidence that illustrates how the inhibition of TGF-ß signaling and the use of oncolytic viruses (OVs) can increase the efficacy of immunotherapy, and discuss the promise and challenges of combining these approaches with immune checkpoint blockade.

The Tango between Cancer-Associated Fibroblasts (CAFs) and Immune Cells in Affecting Immunotherapy Efficacy in Pancreatic Cancer.

The lack of response to therapy in pancreatic ductal adenocarcinoma (PDAC) patients has contributed to PDAC having one of the lowest survival rates of all cancer types. The poor survival of PDAC patients urges the exploration of novel treatment strategies. Immunotherapy has shown promising results in several other cancer types, but it is still ineffective in PDAC. What sets PDAC apart from other cancer types is its tumour microenvironment (TME) with desmoplasia and low immune infiltration and activity. The most abundant cell type in the TME, cancer-associated fibroblasts (CAFs), could be instrumental in why low immunotherapy responses are observed. CAF heterogeneity and interactions with components of the TME is an emerging field of research, where many paths are to be explored. Understanding CAF-immune cell interactions in the TME might pave the way to optimize immunotherapy efficacy for PDAC and related cancers with stromal abundance. In this review, we discuss recent discoveries on the functions and interactions of CAFs and how targeting CAFs might improve immunotherapy.

NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-ß in vitro, although TGF-ß signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.

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.

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.

BDCA3(+)CLEC9A(+) human dendritic cell function and development.

Dendritic cells (DC) are the most potent antigen presenting cells (APC). They comprise a family of different subsets and play an essential role in the induction and regulation of immune responses. Recently, gene expression profiling identified BDCA3(+)CLEC9A(+) DC as a separate human DC subset. This subset was identified in blood, where they represent the smallest population of human DC, as well as in lymphoid and peripheral tissues. This review summarizes the phenotypic, functional and developmental characteristics of BDCA3(+)CLEC9A(+) DC in relation to their mouse equivalents CD8α(+) DC and CD103(+) DC and other human DC subsets. Apart from being potent antigen presenting cells, their specialized functional capacities compared to other human DC subsets, indicate that these BDCA3(+)CLEC9A(+) DC are of major importance in the induction of anti-viral and anti-tumor immunity. Further characterization of their functional properties, developmental pathways and underlying molecular mechanisms may identify target molecules to fully exploit the immune modulatory function of BDCA3(+)CLEC9A(+) DC and potential use of these cells in immunotherapy.

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.

Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells.

Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, we sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-κB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. We have identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection.

FcRγ-chain ITAM signaling is critically required for cross-presentation of soluble antibody-antigen complexes by dendritic cells.

The uptake of Ag-Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ(-/-)) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ(-/-) DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow-derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ(-/-) DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ(-/-) DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4(+) OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.

Kupffer cells interact with hepatitis B surface antigen in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function.

BACKGROUND: Based on their localization, Kupffer cells (KCs) likely interact with hepatitis B virus (HBV). However, the role of KCs in inducing immunity toward HBV is poorly understood. Therefore, the interaction of hepatitis B surface antigen (HBsAg) and KCs, and possible functional consequences, were assessed. METHODS: KCs in liver tissue from patients with chronic HBV were analyzed for presence of HBsAg and their phenotype, and compared with KCs in control liver tissue. Liver graft perfusate-derived KCs and in vitro-generated monocyte-derived macrophages were investigated for functional interaction with patient-derived HBsAg. RESULTS: Intrahepatic KCs were HBsAg positive and more activated than those from control livers. KCs internalized HBsAg in vitro, which did not change their phenotype, but strongly induced proinflammatory cytokine production. Additionally, monocyte-derived macrophages also interacted with HBsAg, leading to activation and cytokine production. Furthermore, HBsAg-exposed macrophages and KC activated natural killer (NK) cells, resulting in increased CD69 expression and interferon-γ production. CONCLUSIONS: KCs directly interact with HBsAg in vivo and in vitro. HBsAg-induced cytokine production by KCs and monocyte-derived macrophages and subsequent NK cell activation may be an early event in viral containment and may support induction of HBV-specific immunity upon HBV infection, but may also contribute to liver pathology.

Fcγ receptor IIb strongly regulates Fcγ receptor-facilitated T cell activation by dendritic cells.

FcγR ligation by Ag-Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates dendritic cell (DC) maturation, a requirement for effective T cell activation. Besides the activating FcγRI, FcγRIII, and FcγRIV, the inhibitory FcγRIIb is expressed on DCs. It is unclear how the ratio between signals from the activating FcγR and the inhibitory FcγRIIb determines the outcome of FcγR ligation on DCs. By microarray analysis, we compared the transcriptomes of steady state and IC-activated bone marrow-derived wild-type (WT) DCs expressing all FcγR or DCs expressing only activating FcγR (FcγRIIb knockout [KO]) or only the inhibitory FcγRIIb (FcR γ-chain KO). In WT DCs, we observed a gene expression profile associated with effective T cell activation, which was absent in FcR γ-chain KO, but strikingly more pronounced in FcγRIIb KO bone marrow-derived DCs. These microarray results, confirmed at the protein level for many cytokines and other immunological relevant genes, demonstrate that the transcriptome of IC-activated DCs is dependent on the presence of the activating FcγR and that the modulation of the expression of the majority of the genes was strongly regulated by FcγRIIb. Our data suggest that FcγRIIb-deficient DCs have an improved capacity to activate naive T lymphocytes. This was confirmed by their enhanced FcγR-dependent Ag presentation and in vivo induction of CD8(+) T cell expansion compared with WT DCs. Our findings underscore the potency of FcγR ligation on DCs for the effective induction of T cell immunity by ICs and the strong regulatory role of FcγRIIb.

Neutralizing Antibodies Impair the Oncolytic Efficacy of Reovirus but Permit Effective Combination with T cell-Based Immunotherapies.

Reovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell-deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell-based immunotherapeutic strategies.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Circulating specific antibodies enhance systemic cross-priming by delivery of complexed antigen to dendritic cells in vivo.

Increasing evidence suggests that antibodies can have stimulatory effects on T-cell immunity. However, the contribution of circulating antigen-specific antibodies on MHC class I cross-priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross-presentation of antigen to CD8(+) T cells. Mice with hapten-specific circulating antibodies, but naϊve for the T-cell antigen, were infused with haptenated antigen and CD8(+) T-cell induction was measured. Mice with circulating hapten-specific antibodies showed significantly enhanced cross-presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross-presentation in mice with circulating antigen-specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c(+) APCs were responsible for the enhanced and sustained cross-presentation, although CD11c(-) APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen-antibody immune complexes improves MHC class I cross-presentation, and CD8(+) T-cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor-associated antigens intensively used in the clinic nowadays.

Preexisting immunity: Barrier or bridge to effective oncolytic virus therapy?

Oncolytic viruses (OVs) represent a highly promising treatment strategy for a wide range of cancers, by mediating both the direct killing of tumor cells as well as mobilization of antitumor immune responses. As many OVs circulate in the human population, preexisting OV-specific immune responses are prevalent. Indeed, neutralizing antibodies (NAbs) are abundantly present in the human population for commonly used OVs, such as Adenovirus type 5 (Ad5), Herpes Simplex Virus-1 (HSV-1), Vaccinia virus, Measles virus, and Reovirus. This review discusses (pre)clinical evidence regarding the effect of preexisting immunity against OVs on two distinct aspects of OV therapy; OV infection and spread, as well as the immune response induced upon OV therapy. Combined, this review provides evidence that consideration of preexisting immunity is crucial in realizing the full potential of the highly promising therapeutic implementation of OVs. Future investigation of current gaps in knowledge highlighted in this review should yield a more complete understanding of this topic, ultimately allowing for better and more personalized OV therapies.

How B cells drive T-cell responses: A key role for cross-presentation of antibody-targeted antigens.

In this review we discuss an underexposed mechanism in the adaptive immune system where B cell and T cell immunity collaborate. The main function of B cell immunity is the generation of antibodies which are well known for their high affinity and antigen-specificity. Antibodies can bind antigens in soluble form making so-called immune complexes (ICs) or can opsonize antigen-exposing cells or particles for degradation. This leads to well-known effector mechanisms complement activation, antibody-dependent cytotoxicity and phagocytosis. What is less realized is that antibodies can play an important role in the targeting of antigen to dendritic cells (DCs) and thereby can drive T cell immunity. Here we summarize the studies that described this highly efficient process of antibody-mediated antigen uptake in DCs in vitro and in vivo. Only very low doses of antigen can be captured by circulating antibodies and subsequently trapped by DCs in vivo. We studied the handling of these ICs by DCs in subcellular detail. Upon immune complex engulfment DCs can sustain MHC class I and II antigen presentation for many days. Cell biological analysis showed that this function is causally related to intracellular antigen-storage compartments which are functional endolysosomal organelles present in DCs. We speculate that this function is immunologically very important as DCs require time to migrate from the site of infection to the draining lymph nodes to activate T cells. The implications of these findings and the consequences for the immune system, immunotherapy with tumor-specific antibodies and novel vaccination strategies are discussed.

Preclinical evaluation of the gorilla-derived HAdV-B AdV-lumc007 oncolytic adenovirus 'GoraVir' for the treatment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune-suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour-specific viruses to eliminate cancerous cells. Non-human primate adenoviruses of the human adenovirus B (HAdV-B) species have demonstrated considerable lytic potential in human cancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla-derived HAdV-B AdV-lumc007 named 'GoraVir'. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic-cancer-associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co-culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC-3 xenograft model at 10 days post-treatment. Collectively, these data demonstrate that the new gorilla-derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour-adjacent stroma.