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.

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.

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.

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC is characterized by a desmoplastic, hypoxic and highly immunosuppressive tumor microenvironment (TME), where T-cell infiltration is often lacking (immune desert), or where T cells are located distant from the tumor islands (immune excluded). Converting the TME to an immune-inflamed state, allowing T-cell infiltration, could increase the success of immunotherapy in PDAC. METHOD: In this study, we use the KPC3 subcutaneous PDAC mouse model to investigate the role of tumor-derived sialic acids in shaping the tumor immune landscape. A sialic acid deficient KPC3 line was generated by genetic knock-out of the CMAS (cytidine monophosphate N-acetylneuraminic acid synthetase) enzyme, a critical enzyme in the synthesis of sialic acid-containing glycans. The effect of sialic acid-deficiency on immunotherapy efficacy was assessed by treatment with anti-programmed cell death protein 1 (PD-1) and agonistic CD40. RESULT: The absence of sialic acids in KPC3 tumors resulted in increased numbers of CD4+ and CD8+ T cells in the TME, and reduced frequencies of CD4+ regulatory T cells (Tregs) within the T-cell population. Importantly, CD8+ T cells were able to infiltrate the tumor islands in sialic acid-deficient tumors. These favorable alterations in the immune landscape sensitized sialic acid-deficient tumors to immunotherapy, which was ineffective in sialic acid-expressing KPC3 tumors. In addition, high expression of sialylation-related genes in human pancreatic cancer correlated with decreased CD8+ T-cell infiltration, increased presence of Tregs, and poorer survival probability. CONCLUSION: Our results demonstrate that tumor-derived sialic acids mediate T-cell exclusion within the PDAC TME, thereby impairing immunotherapy efficacy. Targeting sialic acids represents a potential strategy to enhance T-cell infiltration and improve immunotherapy outcomes in PDAC.

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.

Intertumoral Differences Dictate the Outcome of TGF-ß Blockade on the Efficacy of Viro-Immunotherapy.

The absence of T cells in the tumor microenvironment of solid tumors is a major barrier to cancer immunotherapy efficacy. Oncolytic viruses, including reovirus type 3 Dearing (Reo), can recruit CD8+ T cells to the tumor and thereby enhance the efficacy of immunotherapeutic strategies that depend on high T-cell density, such as CD3-bispecific antibody (bsAb) therapy. TGF-ß signaling might represent another barrier to effective Reo&CD3-bsAb therapy due to its immunoinhibitory characteristics. Here, we investigated the effect of TGF-ß blockade on the antitumor efficacy of Reo&CD3-bsAb therapy in the preclinical pancreatic KPC3 and colon MC38 tumor models, where TGF-ß signaling is active. TGF-ß blockade impaired tumor growth in both KPC3 and MC38 tumors. Furthermore, TGF-ß blockade did not affect reovirus replication in both models and significantly enhanced the Reo-induced T-cell influx in MC38 colon tumors. Reo administration decreased TGF-ß signaling in MC38 tumors but instead increased TGF-ß activity in KPC3 tumors, resulting in the accumulation of α-smooth muscle actin (αSMA+) fibroblasts. In KPC3 tumors, TGF-ß blockade antagonized the antitumor effect of Reo&CD3-bsAb therapy, even though T-cell influx and activity were not impaired. Moreover, genetic loss of TGF-ß signaling in CD8+ T cells had no effect on therapeutic responses. In contrast, TGF-ß blockade significantly improved therapeutic efficacy of Reo&CD3-bsAb in mice bearing MC38 colon tumors, resulting in a 100% complete response. Further understanding of the factors that determine this intertumor dichotomy is required before TGF-ß inhibition can be exploited as part of viroimmunotherapeutic combination strategies to improve their clinical benefit. Significance: Blockade of the pleiotropic molecule TGF-ß can both improve and impair the efficacy of viro-immunotherapy, depending on the tumor model. While TGF-ß blockade antagonized Reo&CD3-bsAb combination therapy in the KPC3 model for pancreatic cancer, it resulted in 100% complete responses in the MC38 colon model. Understanding factors underlying this contrast is required to guide therapeutic application.

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.

Preinduced reovirus-specific T-cell immunity enhances the anticancer efficacy of reovirus therapy.

BACKGROUND: Many solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells. METHODS: We performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells. RESULTS: Reovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth. CONCLUSIONS: These findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.

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.

Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models.

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

Preconditioning of the tumor microenvironment with oncolytic reovirus converts CD3-bispecific antibody treatment into effective immunotherapy.

BACKGROUND: T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. METHODS: The mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. RESULTS: Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease. CONCLUSIONS: Oncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.

Oncolytic virotherapy for pancreatic ductal adenocarcinoma: A glimmer of hope after years of disappointment?

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and highly lethal malignancies. Existing therapeutic interventions have so far been unsuccessful in improving prognosis, and survival remains very poor. Oncolytic virotherapy represents a promising, yet not fully explored, alternative strategy for the treatment of PDAC. Oncolytic viruses (OVs) infect, replicate within and lyse tumor cells specifically and stimulate antitumor immune responses. Multiple challenges have hampered the efficacy of oncolytic virotherapy for PDAC, the most significant being the desmoplastic and immunosuppressive pancreatic tumor microenvironment (TME). The TME limits the access of therapeutic drugs and the infiltration of effector T cells and natural killer (NK) cells into the tumor mass. Additionally, cancer cells promote the secretion of immunosuppressive factors and develop mechanisms to evade the host immune system. Because of their oncolytic and immune-stimulating properties, OVs are the ideal candidates for counteracting the pancreatic immunosuppressive TME and for designing combination therapies that can be clinically exploited in clinical trials that seek to improve the prognosis of PDAC.

Dendritic cell vaccination and CD40-agonist combination therapy licenses T cell-dependent antitumor immunity in a pancreatic carcinoma murine model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. METHODS: Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. RESULTS: Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. CONCLUSION: These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.

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.

Arming oncolytic reovirus with GM-CSF gene to enhance immunity.

Oncolytic reovirus administration has been well tolerated by cancer patients in clinical trials. However, its anti-cancer efficacy as a monotherapy remains to be augmented. We and others have previously demonstrated the feasibility of producing replication-competent reoviruses expressing a heterologous transgene. Here, we describe the production of recombinant reoviruses expressing murine (mm) or human (hs) GM-CSF (rS1-mmGMCSF and rS1-hsGMCSF, respectively). The viruses could be propagated up to 10 passages while deletion mutants occurred only occasionally. In infected cell cultures, the secretion of GM-CSF protein (up to 481 ng/106 cells per day) was demonstrated by ELISA. The secreted mmGM-CSF protein was functional in cell culture, as demonstrated by the capacity to stimulate the survival and proliferation of the GM-CSF-dependent dendritic cell (DC) line D1, and by its ability to generate DCs from murine bone marrow cells. Importantly, in a murine model of pancreatic cancer we found a systemic increase in DC and T-cell activation upon intratumoral administration of rS1-mmGMCSF. These data demonstrate that reoviruses expressing functional GM-CSF can be generated and have the potential to enhance anti-tumor immune responses. The GM-CSF reoviruses represent a promising new agent for use in oncolytic virotherapy strategies.

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.

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.

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.