Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer.

Key Points• Cross-presentation of both soluble and cell-associated tumor antigens by human DC subsets is enhanced by addition of adjuvant TLR agonists. • Ability to cross-present exogenous antigen with high IFN␣ secretion puts human pDCs as activators of CD8 ϩ T cells in antitumor responses. IntroductionDendritic cells (DCs) are the professional antigen presenting cells (APCs) of the immune system with the unique capacity to attract and activate naive CD4 ϩ and CD8 ϩ T cells. 1 After infection or inflammation, DCs undergo a complex maturation process and migrate into lymph nodes where they present antigens (Ags) to T cells. The DC family is very heterogeneous and consists of different DC subsets, each with distinct functional characteristics. In human peripheral blood, at least 2 main populations of DCs can be distinguished: CD11c positive myeloid DCs (mDCs) and CD11c negative plasmacytoid DCs (pDCs). Myeloid DCs can be further subdivided based on the expression of CD16, CD1c, and BDCA3. 2 Transcriptional profiling revealed significant differences between the human blood DC subsets, 3 probably reflecting differences in their Ag-presenting capacities. Furthermore, mDCs and pDCs show clearly different responses to products derived from pathogens, as a result of their distinct Toll-like receptor (TLR) expression profiles. 4 Myeloid DCs have the capacity to produce IL-12 in response to microbial stimuli through TLRs, and thereby, induce Th1 responses. 5,6 Plasmacytoid DCs (pDCs), in contrast, are the key effectors in innate immunity because of their capacity to produce large amounts of type I IFNs in response to bacterial or viral infections. 7 Similar to mDC-derived IL-12, pDC-derived type I IFNs also participate in T-cell priming as Th1-inducing cytokines. 8 In addition to directing CD4 ϩ Th responses, DCs are also important for the generation of CD8 ϩ cytotoxic T-cell responses against viruses and tumors. As professional APCs, DCs have the unique capacity to take up, process, and present exogenously encountered Ags for cross-presentation via MHC class I molecules to CD8 ϩ T cells. Numerous studies have been performed to comprehend this cross-presentation process, and these have revealed 2 major pathways: (1) the "canonical" proteasome dependent cytosolic pathway, and (2) the TAP and proteasome independent pathway. [9][10][11][12] Many studies however, made use of murine DCs to study cross-presentation capacities and mechanisms used by different DC subsets. There is ample evidence that identified the CD8␣ ϩ DC as the superior cross-presenting DC subset in mice. 13,14 Recently, a lot of effort has been put toward finding the human counterpart of the murine cross-presenting CD8␣ ϩ DC subset. Despite basic similarities between human and mouse DCs, direct comparison is difficult because of large differences in cell-surface markers and TLR expression, in particular also for pDCs, which in contrast to mice are the sole TLR9-expressing subtype of DCs in Submitted June 7, 2012; accepted November 9, 2012. Prepublished onl...

The tumor microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1þ CD14 þ population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell-based vaccines, where they suppress CD4 þ T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1 þ

Dendritic cells (DCs) are central in maintaining the intricate balance between immunity and tolerance by orchestrating adaptive immune responses. Being the most potent antigen presenting cells, DCs are capable of educating naïve T cells into a wide variety of effector cells ranging from immunogenic CD4+ T helper cells and cytotoxic CD8+ T cells to tolerogenic regulatory T cells. This education is based on three fundamental signals. Signal I, which is mediated by antigen/major histocompatibility complexes binding to antigen-specific T cell receptors, guarantees antigen specificity. The co-stimulatory signal II, mediated by B7 family molecules, is crucial for the expansion of the antigen-specific T cells. The final step is T cell polarization by signal III, which is conveyed by DC-derived cytokines and determines the effector functions of the emerging T cell. Although co-stimulation is widely recognized to result from the engagement of T cell-derived CD28 with DC-expressed B7 molecules (CD80/CD86), other co-stimulatory pathways have been identified. These pathways can be divided into two groups based on their impact on primed T cells. Whereas pathways delivering activatory signals to T cells are termed co-stimulatory pathways, pathways delivering tolerogenic signals to T cells are termed co-inhibitory pathways. In this review, we discuss how the nature of DC-derived signal II determines the quality of ensuing T cell responses and eventually promoting either immunity or tolerance. A thorough understanding of this process is instrumental in determining the underlying mechanism of disorders demonstrating distorted immunity/tolerance balance, and would help innovating new therapeutic approaches for such disorders.

Plasmacytoid dendritic cells (pDCs) play a crucial role in initiating immune responses by secreting large amounts of type I IFNs. Currently, the role for human pDCs as professional APCs in the cross-presentation of exogenous Ags is being re-evaluated. Human pDCs are equipped with a broad repertoire of Ag uptake receptors and an efficient Ag-processing machinery. In this study, we set out to investigate which receptor can best be deployed to deliver Ag to pDCs for Ag (cross-)presentation. We show that targeting nanoparticles to pDCs via the C-type lectins DEC-205, DC immunoreceptor, blood DC Ag-2, or the FcR CD32 led to uptake, processing, and (cross-) presentation of encapsulated Ag to both CD4+ and CD8+ T cells. This makes these receptors good candidates for potential in vivo targeting of pDCs by nanocarriers. Notably, the coencapsulated TLR7 agonist R848 efficiently activated pDCs, resulting in phenotypical maturation as well as robust IFN-α and TNF-α production. Taken together, their cross-presentation capacity and type I IFN production to further activate components of both the innate and adaptive immune system mark pDCs as inducers of potent antitumor responses. These findings pave the way to actively recruit human pDCs for cellular cancer immunotherapy.

c PI4KIII␤ recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIII␤ to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIII␤ to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although HRV 3A was previously shown to interact with ACBD3, our data suggest that PI4KIII␤ recruitment occurred independently of both GBF1 and ACBD3. E nteroviruses (family Picornaviridae), such as poliovirus (PV) and coxsackievirus B3 (CVB3), rely on host factor phosphatidylinositol-4-kinase III␤ (PI4KIII␤) for genome replication (1, 2). The recruitment of PI4KIII␤ to the replication sites is mediated by the 3A viral nonstructural protein (1). PI4KIII␤ is normally recruited to the Golgi membranes by the small GTPase Arf1 (3) and the Arf1 activator guanine nucleotide exchange factor (GEF) GBF1 (4). ACBD3 (acyl-coenzyme A [CoA]-binding protein domain 3) also interacts with PI4KIII␤ to recruit it to the Golgi membranes (5, 6). CVB3 3A and PV 3A both interact with the N terminus of GBF1 (7, 8) as well as ACBD3 (9). However, we and others recently showed that the PI4KIII␤ recruitment by 3A of PV and CVB3 seems to occur independently of both GBF1/Arf1 and ACBD3 (9, 10). We studied this by individually expressing mutant 3A proteins of CVB3 that no longer interact with GBF1 (9). Unfortunately, these 3A mutations render CVB3 unviable (F. J. M. van Kuppeveld, unpublished data); thus, we have not yet been able to study the role of GBF1 in PI4KIII␤ recruitment in infected cells. Yeast two-hybrid analysis suggests that the 3A proteins of human rhinoviruses (HRV), which also belong to the Enterovirus genus, do not interact with the N terminus of GBF1 (8). In this study, we set out to investigate the consequences of the inability of HRV 3A proteins to interact with GBF1, focusing on the possible role of GBF1 and ACBD3 in PI4KIII␤ recruitment and virus replication.First, we studied the interaction between the N terminus of GBF1 and the 3A proteins of HRV2 and HRV14, which are members of group A and B rhinoviruses, respectively, in a mammalian environment, employing the mammalian two-hybrid (M2H) assay (Promega) as described previously (9). We used the 3As of CVB3, PV, and mengovirus, belonging to the Cardiovirus genus of the Picornaviridae, as controls. Expression of all 3A proteins was verified by Western blot analysis (Fig. 1A, lower half). Importantly, all enterovirus 3A proteins were previously tested and validated to be competent in interacting with other proteins in the M2H assay (9). Both CVB3 3A and PV 3A interacted with the N terminus of GBF1, while no interaction was detected for mengovirus 3A (Fig. 1A, upper panel). HRV2 3A did not bind the N terminus of GBF1, while HRV14 3A interacted to only a limited extent. We considered the possibility that the HRV 3A proteins bind to the C terminus of GBF1. Therefore, we studied whether 3A expression induced recruitment of full-length...

Dendritic cells (DCs) are key in connecting innate and adaptive immunity. Their potential in inducing specific immune responses has made them interesting targets for immunotherapeutic approaches. Our research group was the first to exploit the naturally occurring myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in therapeutic vaccination trials against melanoma. To develop primary DC subsets as an optimal vaccine, the identification of a clinically applicable adjuvant activating both subsets is required. Although the expression of pathogen recognition receptors differs distinctly between the DC subsets, both pDCs and mDCs can respond to single-stranded RNA (ssRNA) via Toll-like receptors 7 and 8, respectively. Since ssRNA is easily degraded by RNases, we stabilized anionic RNA by complexing it with the positively charged protein protamine. This leads to the formation of protamine–RNA complexes with varying features depending on ionic content. We subsequently investigated the immunostimulatory effect of complexes that formed various salt concentrations on purified DC subsets. Both mDCs and pDCs upregulated maturation markers and produced pro-inflammatory cytokines in a dose-dependent way to the protamine–RNA complexes. This was dependent on endosomal acidification and correlated partly with the uptake of protamine–RNA complexes. Furthermore, both DC subsets induced T cell proliferation and IFN gamma secretion in a beneficial ratio to IL-10. These results indicate that protamine–RNA complexes can be used to stimulate human mDC and pDC ex vivo for use in immunotherapeutic settings.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1746-9) contains supplementary material, which is available to authorized users.

There has recently been a paradigm shift in the field of dendritic cell (DC)-based immunotherapy, where several clinical studies have confirmed the feasibility and advantageousness of using directly isolated human blood-derived DCs over in vitro differentiated subsets. There are two major DC subsets found in blood; plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), and both have been tested clinically. CD1c+ mDCs are highly efficient antigen-presenting cells that have the ability to secrete IL-12p70, while pDCs are professional IFN-α-secreting cells that are shown to induce innate immune responses in melanoma patients. Hence, combining mDCs and pDCs poses as an attractive, multi-functional vaccine approach. However, type I IFNs have been reported to inhibit IL-12p70 production and mDC-induced T-cell activation. In this study, we investigate the effect of IFN-α on mDC maturation and function. We demonstrate that both recombinant IFN-α and activated pDCs strongly enhance mDC maturation and increase IL-12p70 production. Co-cultured mDCs and pDCs additionally have beneficial effect on NK and NKT-cell activation and also enhances IFN-γ production by allogeneic T cells. In contrast, the presence of type I IFNs reduces the proliferative T-cell response. The mere presence of a small fraction of activated pDCs is sufficient for these effects and the required ratio between the subsets is non-stringent. Taken together, these results support the usage of mDCs and pDCs combined into one immunotherapeutic vaccine with broad immunostimulatory features.Electronic supplementary materialThe online version of this article (10.1007/s00262-018-2204-2) contains supplementary material, which is available to authorized users.