Dendritic cells (DCs) are crucial for priming of naive CD8 ⁺ T lymphocytes to exogenous antigens, so-called “cross-priming.” We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I–peptide complexes at the cell surface.

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.

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.

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 bothin vitroand in sera of HBV-infected patients. The abundance of sCD14-HBsAg complexes varied between chronic HBV disease stages and correlated with activation of BDCA1⁺mDCin 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.IMPORTANCEHepatitis 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.

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.