Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming.

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.

Naturally produced type I IFNs enhance human myeloid dendritic cell maturation and IL-12p70 production and mediate elevated effector functions in innate and adaptive immune cells.

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.

Expansion of a BDCA1+CD14+ Myeloid Cell Population in Melanoma Patients May Attenuate the Efficacy of Dendritic Cell Vaccines.

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 BDCA1(+)CD14(+) population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell-based vaccines, where they suppress CD4(+) T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1(+)CD14(+) cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1(+)CD14(+) cell population expressed markers of both BDCA1(+) dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumors for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy. Cancer Res; 76(15); 4332-46. ©2016 AACR.

A Comparative Study of the T Cell Stimulatory and Polarizing Capacity of Human Primary Blood Dendritic Cell Subsets.

Dendritic cells (DCs) are central players of immune responses; they become activated upon infection or inflammation and migrate to lymph nodes, where they can initiate an antigen-specific immune response by activating naive T cells. Two major types of naturally occurring DCs circulate in peripheral blood, namely, myeloid and plasmacytoid DCs (pDCs). Myeloid DCs (mDCs) can be subdivided based on the expression of either CD1c or CD141. These human DC subsets differ in surface marker expression, Toll-like receptor (TLR) repertoire, and transcriptional profile, suggesting functional differences between them. Here, we directly compared the capacity of human blood mDCs and pDCs to activate and polarize CD4(+) T cells. CD141(+) mDCs show an overall more mature phenotype over CD1c(+) mDC and pDCs; they produce less IL-10 and more IL-12 than CD1c(+) mDCs. Despite these differences, all subsets can induce the production of IFN-γ in naive CD4(+) T cells. CD1c(+) and CD141(+) mDCs especially induce a strong T helper 1 profile. Importantly, naive CD4(+) T cells are not polarized towards regulatory T cells by any subset. These findings further establish all three human blood DCs-despite their differences-as promising candidates for immunostimulatory effectors in cancer immunotherapy.

Lipid peroxidation causes endosomal antigen release for cross-presentation.

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.

Protamine-stabilized RNA as an ex vivo stimulant of primary human dendritic cell subsets.

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.

Primary Human Blood Dendritic Cells for Cancer Immunotherapy-Tailoring the Immune Response by Dendritic Cell Maturation.

Dendritic cell (DC)-based cancer vaccines hold the great promise of tipping the balance from tolerance of the tumor to rejection. In the last two decades, we have gained tremendous knowledge about DC-based cancer vaccines. The maturation of DCs has proven indispensable to induce immunogenic T cell responses. We review the insights gained from the development of maturation cocktails in monocyte derived DC-based trials. More recently, we have also gained insights into the functional specialization of primary human blood DC subsets. In peripheral human blood, we can distinguish at least three primary DC subsets, namely CD1c⁺ and CD141⁺ myeloid DCs and plasmacytoid DCs. We reflect the current knowledge on maturation and T helper polarization by these blood DC subsets in the context of DC-based cancer vaccines. The maturation stimulus in combination with the DC subset will determine the type of T cell response that is induced. First trials with these natural DCs underline their excellent in vivo functioning and mark them as promising tools for future vaccination strategies.

GBF1- and ACBD3-independent recruitment of PI4KIIIß to replication sites by rhinovirus 3A proteins.

PI4KIIIß recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIIIß to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIß 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 PI4KIIIß recruitment occurred independently of both GBF1 and ACBD3.

Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes.

T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were in the range of 1-17 (median = 5), and in several patients, the number of clonotypes expanded within tumor lesions resembled that observed among autologous peripheral blood mononuclear cells. Moreover, several of these clonotypes were identical in TILs and PBMCs. Flow cytometry data demonstrated that the general differentiation status of CD8+ TILs differed from that of circulating CD8+ T cells. Furthermore, PD-1 and LAG-3 were expressed by a significantly higher percentage of CD8+ RCC-infiltrating lymphocytes as compared with PBMCs obtained from RCC patients or healthy individuals. Thus, CD8+ TILs display a differentiated phenotype and express activation markers as well as surface molecules associated with the inhibition of T-cell functions. However, TILs are characterized by a low amount of expanded T-cell clonotypes.

Targeting uptake receptors on human plasmacytoid dendritic cells triggers antigen cross-presentation and robust type I IFN secretion.

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.

The nature of activatory and tolerogenic dendritic cell-derived signal II.

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.

Human plasmacytoid dendritic cells efficiently cross-present exogenous Ags to CD8+ T cells despite lower Ag uptake than myeloid dendritic cell subsets.

In human peripheral blood, 4 populations of dendritic cells (DCs) can be distinguished, plasmacytoid dendritic cells (pDCs) and CD16(+), CD1c(+), and BDCA-3(+) myeloid DCs (mDCs), each with distinct functional characteristics. DCs have the unique capacity to cross-present exogenously encountered antigens (Ags) to CD8(+) T cells. Here we studied the ability of all 4 blood DC subsets to take up, process, and present tumor Ags to T cells. Although pDCs take up less Ags than CD1c(+) and BDCA3(+) mDCs, pDCs induce potent Ag-specific CD4(+) and CD8(+) T-cell responses. We show that pDCs can preserve Ags for prolonged periods of time and on stimulation show strong induction of both MHC class I and II, which explains their efficient activation of both CD4(+) and CD8(+) T cells. Furthermore, pDCs cross-present soluble and cell-associated tumor Ags to cytotoxic T lymphocytes equally well as BDCA3(+) mDCs. These findings, and the fact that pDCs outnumber BDCA3(+) mDCs, both in peripheral blood and lymph nodes, together with their potent IFN-I production, known to activate both components of the innate and adaptive immune system, put human pDCs forward as potent activators of CD8(+) T cells in antitumor responses. Our findings may therefore have important consequences for the development of antitumor immunotherapy.