Immunogenic DCs Research Articles

Cholesterol controls tolerogenic versus immunogenic DCs.

Cholesterol controls tolerogenic versus immunogenic DCs.

Tumor-associated human dendritic cell subsets: Phenotype, functional orientation, and clinical relevance.

DCs play a pivotal role in orchestrating innate and adaptive antitumor immunity. Activated DCs can produce large amounts of various proinflammatory cytokines, initiate T-cell responses, and exhibit direct cytotoxicity against tumor cells. They also efficiently enhance the antitumoral properties of NK cells and T lymphocytes. Based on these capabilities, immunogenic DCs promote tumor elimination and are associated with improved survival of patients. Furthermore, they can essentially contribute to the clinical efficacy of immunotherapeutic strategies for cancer patients. However, depending on their intrinsic properties and the tumor microenvironment, DCs can be rendered dysfunctional and mediate tolerance by producing immunosuppressive cytokines and activating Treg cells. Such tolerogenic DCs can foster tumor progression and are linked to poor prognosis of patients. Here, we focus on recent studies exploring the phenotype, functional orientation, and clinical relevance of tumor-infiltrating conventional DC1, conventional DC2, plasmacytoid DCs, and monocyte-derived DCs in translational and clinical settings. In addition, recent findings demonstrating the influence of DCs on the efficacy of immunotherapeutic strategies are summarized.

A role for TNF-α in alveolar macrophage damage-associated molecular pattern release.

Chronic beryllium disease (CBD) is a metal hypersensitivity/autoimmune disease in which damage-associated molecular patterns (DAMPs) promote a break in T cell tolerance and expansion of Be2+/self-peptide-reactive CD4+ T cells. In this study, we investigated the mechanism of cell death induced by beryllium particles in alveolar macrophages (AMs) and its impact on DAMP release. We found that phagocytosis of Be led to AM cell death independent of caspase, receptor-interacting protein kinases 1 and 3, or ROS activity. Before cell death, Be-exposed AMs secreted TNF-α that boosted intracellular stores of IL-1α followed by caspase-8-dependent fragmentation of DNA. IL-1α and nucleosomal DNA were subsequently released from AMs upon loss of plasma membrane integrity. In contrast, necrotic AMs released only unfragmented DNA and necroptotic AMs released only IL-1α. In mice exposed to Be, TNF-α promoted release of DAMPs and was required for the mobilization of immunogenic DCs, the expansion of Be-reactive CD4+ T cells, and pulmonary inflammation in a mouse model of CBD. Thus, early autocrine effects of particle-induced TNF-α on AMs led to a break in peripheral tolerance. This potentially novel mechanism may underlie the known relationship between fine particle inhalation, TNF-α, and loss of peripheral tolerance in T cell-mediated autoimmune disease and hypersensitivities.

Induced pluripotent stem cells reprogrammed from primary dendritic cells provide an abundant source of immunostimulatory dendritic cells for use in immunotherapy.

Cell types differentiated from induced pluripotent stem cells (iPSCs) are frequently arrested in their development program, more closely resembling a fetal rather than an adult phenotype, potentially limiting their utility for downstream clinical applications. The fetal phenotype of iPSC‐derived dendritic cells (ipDCs) is evidenced by their low expression of MHC class II and costimulatory molecules, impaired secretion of IL‐12, and poor responsiveness to conventional maturation stimuli, undermining their use for applications such as immune‐oncology. Given that iPSCs display an epigenetic memory of the cell type from which they were originally derived, we investigated the feasibility of reprogramming adult DCs to pluripotency to determine the impact on the phenotype and function of ipDCs differentiated from them. Using murine bone marrow‐derived DCs (bmDCs) as proof of principle, we show here that immature DCs are tractable candidates for reprogramming using non‐integrating Sendai virus for the delivery of Oct4, Sox2, Klf4, and c‐Myc transcription factors. Reprogramming efficiency of DCs was lower than mouse embryonic fibroblasts (MEFs) and highly dependent on their maturation status. Although control iPSCs derived from conventional MEFs yielded DCs that displayed a predictable fetal phenotype and impaired immunostimulatory capacity in vitro and in vivo, DCs differentiated from DC‐derived iPSCs exhibited a surface phenotype, immunostimulatory capacity, and responsiveness to maturation stimuli indistinguishable from the source DCs, a phenotype that was retained for 15 passages of the parent iPSCs. Our results suggest that the epigenetic memory of iPSCs may be productively exploited for the generation of potently immunogenic DCs for immunotherapeutic applications.

IL-33 induces a metabolic programming supporting dendritic cells tolerogenicity

Abstract Objective Delivery of the stromal cell-derived cytokine IL-33 protects allografts from rejection by acting on CD11c+ DC that facilitate the expansion of regulatory T cells (Treg). IL-33 administration also permits tolerance induction at doses of co-stimulatory blockade that fails on their own. Based on these data we hypothesized that IL-33-stimulated DC represent a tolerogenic DC subset and their precise examination will define biochemical pathways that contribute to tolerogenic DC functions. Methods Mouse bone marrow-derived CD11c+ DCs were treated with lipopolysaccharide (LPS) or IL-33 before nitric oxide (NO) generation, glucose and fatty acid uptake, and mitochondrial function and potential were assessed by EPR spectroscopy, flow cytometry, and Seahorse analysis. Results DC responded to the TLR4 ligand LPS by generating high levels of NO and shifting their metabolic activity to aerobic glycolysis. This was in stark contrast to IL-33, which caused a metabolic reprogramming in DC that involved augmented mitochondrial basal respiration, ATP production, and maximum respiration. IL-33-stimulated DC also displayed increase mitochondrial potential and a profound increase in the uptake of fatty acids. Conclusions It has emerged that immunogenic DCs undergo metabolic changes that enables their pro-inflammatory functions. The metabolic underpinnings of tolerogenic DCs is less understood. Here we show that IL-33-stimulated DC, which potently expand Treg, rely on oxidative phosphorylation and uptake fatty acids. These data suggest IL-33 initiates metabolic reprogramming matching those found in regulatory myeloid populations, including tumor associated DC and alternatively activated macrophages.

Knockdown of circular RNA FSCN1 impairs dendritic cell immune function through regulating the NFKB and Foxo3 signaling pathways

Abstract Circular RNAs (circRNAs) that are a new class of endogenously expressed non-coding RNAs produced from back splicing with a covalently closed loop structure have been emerging as an important gene regulator in the physiological and pathological development of cells. It remains unknown about roles of circRNAs in DCs. The objective of the study is to investigate the impact of circular RNA FSCN1 (circFSCN1) on dendritic cell immune function. Bone marrow derived dendritic cells were cultured in vitro and circRNA expression was detected by circRNA microarrays and qRT-PCR. The effect of circFSCN1 on DCs was studied. We found that the expression profiles of circRNAs were significantly different in mature immunogenic DCs v.s immature immunosuppressive DCs. circFSCN1 was the most significantly highly expressed in mature DCs compared with immature DCs. Treatment with immunosuppressive cytokines TGF β and GDF15 reduced circFSCN1 expression in DCs. Knockdown of circFSCN1 using siRNA reduced the phosphorylation of Rel Ap65, but increased phosphorylated Foxo3. Silencing of circFSCN1 impaired DCs to activate T cells, changed inflammatory cytokine production and enhanced Treg generation whereas circFSCN1 siRNA did not affect DC maturation. In conclusion, it is a first report to demonstrate that circFSCN1 is essential for DC immune response.

Abstract 4695: Murine tumor growth suppression through CD8+CTLs via activated DEC-205+dendritic cells by sequential administration of α-galactosylceramide in vivo

Abstract Cancer immunotherapy is mediated by the effective priming and activation of tumor-specific class I major histocompatibility complex molecule-restricted CD8+ cytotoxic T lymphocytes. DEC-205+ dendritic cells can cross-present the epitopes of captured tumor antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumor-specific CD8+ CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of insufficient CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and do not prime CD8+ CTLs. Here, we show that the growing of tumors was suppressed by activated CD8+ CTLs with tumor-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide, which is a compound known to stimulate invariant natural killer T cells and selectively activate DEC-205+ DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation of α-GalCer every 48 hour appeared to convert tolerogenic DEC-205+ DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a greater cross-presentation capacity, which primed CTL precursors and induced tumor-specific CD8+ CTLs within the tumor environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205+ DCs within tumors into immunogenic DCs via the sequential administration of an immuno-potent lipid/glycolipid, and activated immunogenic DCs expressing sufficient suitable co-stimulatory molecules will prime and activate tumor-specific CD8+ CTLs within the tumor to control tumor growing. Citation Format: Hideki Kogo, Hidemi Takahashi, Eiji Uchida. Murine tumor growth suppression through CD8+CTLs via activated DEC-205+dendritic cells by sequential administration of α-galactosylceramide in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4695.

Activation of host p53 enhances anti-tumor immunity by driving differentiation of immunogenic myeloid DCs in tumors, while simultaneously protecting against collateral autoimmunity against shared self-tumor antigens.

Abstract During immunotherapy, we show that large numbers of immunogenic CD103+ DCs can arise in murine tumors via direct differentiation myeloid-lineage (Ly6c+ monocytic) precursors. These “dual-phenotype” Ly6c+CD103+ DCs differentiate from a precursor population of monocytic myeloid-derived suppressor cells (MDSCs) in tumors. Differentiation was controlled by inflammation-induced activation of the transcription factor p53 in MDSCs. Targeted deletion of myeloid p53 selectively ablated the Ly6c+CD103+ DC population, which resulted in abrogation of all response to multiple forms of tumor immunotherapy. Pharmacologically elevating the level of p53 expression in host cells, by administering the p53-agonist (MDM2-inhibitor) drug nutlin-3a, caused a sustained increase in Ly6c+CD103+ DCs in tumors during immunotherapy, and markedly enhanced anti-tumor immunity. Conversely, however, when a nominal tumor antigen (OVA) was also shared by a critical host tissue (Rip-mOVA mice), elevation of host p53 using nutlin-3a was potent immunosuppressive and protective against lethal autoimmunity outside of the tumor, while still driving enhanced immunity – against the same antigen – within the tumor itself. Thus, p53-agonist drugs may represent a novel class immunotherapy agents, with high discrimination between tumor-associated antigens and collateral autoimmunity.

Up-regulation of EP2 and EP3 receptors in human tolerogenic dendritic cells boosts the immunosuppressive activity of PGE2.

Dendritic cells (DCs) are APCs essential in regulating the immune response. PGE2, produced during inflammation, has a pivotal role in the maturation of DCs and, therefore, is vital for the immune response. The large variety of biologic functions governed by PGE2 is mediated by its signaling through 4 distinct E-type prostanoid (EP) receptors. Immunogenic DCs express EP2 and EP4, which mediate the PGE2 signaling. However, the expression and function of EP receptors in human tolerogenic DCs (tol-DCs), which present an inhibitory phenotype, have not yet, to our knowledge, been assessed. To clarify the role of EP receptors in tol-DCs, we examined the expression of different EP receptors and their effect using selective agonists in human cells. We find that EP2 and EP3 expression are up-regulated in in vitro-generated tol-DCs compared with mature DCs (mDCs). Activation of EP2-EP4 has a direct effect on the surface expression of costimulatory molecules and maturation receptors, such as CD80, CD83, and CD86 or MHCII and CCR7 in tol-DCs, the latter being exclusively modulated by PGE2-EP4 signaling. Importantly, we find that EP2 and EP3 receptors are involved in tolerance induction through IL-10 production by tol-DCs. These results are in sharp contrast with the inflammatory role of EP4 Moreover, we show that DCs generated in the presence of agonists for EP receptors, induce naive T cell differentiation toward polarized Th1/Th17 cells. Given the differential effects of EP receptors, our results suggest that EP receptor agonist/antagonists might become relevant novel drug templates to modulate immune response.

Exploiting tumor-associated dendritic cell heterogeneity for novel cancer therapies.

Dendritic cells (DCs) are specialized APCs present in all tissues, including tumors. They play a major role in orchestrating immune responses and were shown to occur in various functional states in tumors. In this respect, immunogenic tumor-associated DCs (TADCs) are required to initiate and sustain T cell-dependent anti-cancer immunity, whereas regulatory TADCs harbor robust immunosuppressive potential and accelerate malignant growth. Importantly, the heterogeneity of the DC compartment in tumors has been dissected recently in murine and human cancers and was shown to consist of developmentally distinct subsets, including conventional DC (cDC)1, cDC2, and monocyte-derived DCs (Mo-DCs). TADCs constitute an essential target in efforts to generate therapeutic immunity against cancer, and the understanding of the complexity of the TADC heterogeneity might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors. Hence, this review addresses the differential functional specializations of ontogenically distinct TADC subsets.

Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis.

Multiple sclerosis (MS) is caused by immune-mediated damage of myelin sheath. Current therapies aim to block such immune responses. However, this blocking is not sufficiently specific and hence compromises immunity, leading to severe side effects. In addition, blocking medications usually provide transient effects and require frequent administration, which further increases the chance to compromise immunity. In this regard, myelin-specific therapy may provide the desired specificity and a long-lasting therapeutic effect by inducing myelin-specific regulatory T (Treg) cells. Tolerogenic dendritic cells (TolDCs) are one such therapy. However, ex vivo generated TolDCs may be converted into immunogenic DCs in a proinflammatory environment. In this study, we identified a potential novel myelin-specific therapy that works with immunogenic DCs, hence without the in vivo conversion concern. We showed that immunization with DCs, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase for de novo synthesis of a focally high 1,25-dihydroxyvitamin D concentration in the peripheral lymphoid tissues, induced Treg cells. In addition, such engineered DCs, when pulsed with a myelin antigen, led to myelin-specific suppression of ongoing experimental allergic encephalomyelitis (an MS animal model), and the disease suppression depended on forkhead-box-protein-P3(foxp3)+ Treg cells. Our data support a novel concept that immunogenic DCs can be engineered for myelin-specific therapy for MS.—Li, C.-H., Zhang, J., Baylink, D. J., Wang, X., Goparaju, N. B., Xu, Y., Wasnik, S., Cheng, Y., Berumen, E. C., Qin, X., Lau, K.-H. W., Tang, X. Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1α-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis.

Tolerogenic dendritic cells show gene expression profiles that are different from those of immunogenic dendritic cells in DBA/1 mice

Tolerogenic dendritic cells (tDCs) play an important role in inducing peripheral tolerance; however, few tDC-specific markers have been identified. The aims of this study were to examine whether tDCs show a different gene expression profile from that of immunogenic DCs and identify specific gene markers of each cell type, in DBA/1 mice. tDCs were generated by treating immature DCs (imDCs) with TNF-α and type II collagen. The gene expression profiles of mature (m)DCs and tDCs were then investigated by microarray analysis and candidate markers were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Supervised selection identified 75 gene signatures, 63 of which were consistently upregulated in mDCs and 12 of which were upregulated only in tDCs. Additionally, 10 genes were overexpressed or equally expressed in both tDCs and mDCs. Scin (tDC-specific genes) and Orm1, Pdlim4 and Enpp2 (mDC-specific genes) were validated by real-time qRT-PCR. Taken together, these results clearly show that tDCs and mDCs can be identified according to their expression of specific gene markers.

Differential effects of monophosphoryl lipid A and cytokine cocktail as maturation stimuli of immunogenic and tolerogenic dendritic cells for immunotherapy

Immunotherapy using monocyte-derived dendritic cells (MDDC) is increasingly being considered as alternative therapeutic approach in cancer, infectious diseases and also in autoimmunity when patients are not responsive to conventional treatments. In general, generation of MDDC from monocytes is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the culture to obtain mature DCs suitable for therapy. For DC maturation, different combinations of pro-inflammatory mediators and Toll-like receptor ligands have been tested, obtaining DCs that differ in their properties and the type of immune response they promote. Therefore, it is necessary to find an optimal cytokine environment for DC maturation to obtain a cellular product suitable for DC-based immunotherapeutic protocols. In this study, we have evaluated in vitro the effects of different maturation stimuli on the viability, phenotype, cytokine profile, stability and functionality of immunogenic and tolerogenic (1α,25-dihydroxyvitamin D(3)-treated) MDDC. Maturation was induced using the clinical grade TLR4-agonist: monophosphoryl lipid A (LA), compared to the traditional cytokine cocktail (CC; clinical grade TNF-α, IL-1β, PGE2) and a combination of both. Our results showed the combination of CC+LA rendered a potent immunogenic DC population that induced the production of IFN-γ and IL-17 in allogeneic co-cultures, suggesting a Th17 polarization. Moreover, these immunogenic DCs showed a high surface expression of CD83, CD86, HLA-DR and secretion of IL-12p70. When aiming to induce tolerance, using LA to generate mature TolDC did not represent a clear advantage, and the stability and the suppressive capability exhibited by CC-matured TolDC may represent the best option. Altogether, these findings demonstrate the relevance of an appropriate maturation stimulus to rationally modulate the therapeutic potential of DCs in immunotherapy.

Cimetidine augments Th1/Th2 dual polarized immune responses to recombinant HBV antigens

Cimetidine (CIM) is a histamine H2 receptor inverse agonist used primarily as an anti-stomach acids drug, but recent studies showed that it may also modulate immune responses. To evaluate its potential usefulness as an adjuvant, we determined its immune modulating effects on subunit immunization using an HBV-derived recombinant protein antigen rHBsAg. CIM activated the PI3K-Akt signaling pathway in DCs. As an adjuvant, it activated immunogenic DCs, deactivated tolerogenic T cells (nTreg), and augmented both Th1- and Th2-polarlized immune responses to rHBsAg. As a result, it enhanced both antibody- and cytotoxic T cell-mediated immune responses. Importantly, in comparison with the FDA-approved human adjuvant alum, CIM is superior in its ability to block IL-10 up-regulation and potentiate Th1/Th2 dual polarization. These results suggest that CIM may be a better adjuvant for therapeutic vaccines against chronic viral infection, such as the HBV infection, where dual polarization should allow more effective elimination of infected host cells.

IL-10 induces aberrant deletion of dendritic cells by natural killer cells in the context of HIV infection

Persistent levels of IL-10 play a central role in progressive immune dysfunction associated with chronic viral infections such as HIV, but the underlying mechanisms are poorly understood. Because IL-10 affects the phenotypic and functional properties of DCs, which are responsible for initiating adaptive immune responses, we investigated whether IL-10 induces changes in DC phenotype and function in the context of HIV infection. Here, we show that IL-10 treatment of immature and mature human DCs in culture induced contrasting phenotypic changes in these populations: immature DCs exhibited aberrant resistance to NK cell-mediated elimination, whereas mature DCs exhibited increased susceptibility to NKG2D-dependent NK elimination. Treatment of immature and mature DCs with HIV resulted in potent IL-10 secretion and the same phenotypic and functional changes observed in the IL-10-treated cells. Consistent with these in vitro data, LNs isolated from individuals infected with HIV exhibited aberrant accumulation of a partially "immature" DC population. Together, these data suggest that the progressive immune dysfunction observed in chronic viral infections might be caused in part by IL-10-induced reversal of DC susceptibility to NK cell-mediated elimination, resulting in the accumulation of poorly immunogenic DCs in LNs, the sites of adaptive immune response induction.

Activated Inflammatory Infiltrate in HSV-1-Infected Corneas without Herpes Stromal Keratitis

To investigate herpes stromal keratitis (HSK) immunopathology by studying HSV-1-infected corneas that fail to develop HSK. Plaque assay quantified HSV-1 in the tear film of infected mice. FACS analysis enumerated corneal leukocytic infiltrate and characterized infiltrate phenotypically after staining for activation and regulatory T cell (Treg) markers and for markers of antigen-presenting cell (APC) maturation. Treg cells were depleted in vivo using anti-CD25 mAb. Luminex analysis quantified the amount of cytokines and chemokines expressed in corneal tissue homogenate. Infected corneas without HSK exhibited a pronounced leukocytic infiltrate containing a significantly higher proportion and nearly identical absolute number of activated CD4+ T cells 15 days after infection when compared with those with HSK. Moreover, the frequency and absolute number of regulatory CD4+ T cells (Tregs) was lower in nondiseased corneas, and Treg depletion did not influence HSK incidence. The frequency of mature, immunogenic DCs and the ratio of mature DCs to CD4+ T cells were nearly identical in corneas with and without HSK. The authors observed a reduced population of neutrophils and reduced expression of neutrophil chemoattractants MIP-1beta and keratinocyte chemoattractant and the neutrophil-attracting cytokine IL-6 in corneas without HSK. These findings demonstrate that HSV-1-infected corneas can retain clarity in the presence of a substantial secondary leukocytic infiltrate, that activated CD4+ T cells, while necessary, are not sufficient for HSK development, that susceptibility to HSK is not determined by Tregs, and that clinical disease correlates with the accumulation of a critical mass of neutrophils through chemoattraction.

Kynurenine Pathway Enzymes in Dendritic Cells Initiate Tolerogenesis in the Absence of Functional IDO

Dendritic cell (DC) tryptophan catabolism has emerged in recent years as a major mechanism of peripheral tolerance. However, there are features of this mechanism, initiated by IDO, that are still unclear, including the role of enzymes that are downstream of IDO in the kynurenine pathway and the role of the associated production of kynurenines. In this study, we provide evidence that 1) murine DCs express all enzymes necessary for synthesis of the downstream product of tryptophan breakdown, quinolinate; 2) IFN-gamma enhances transcriptional expression of all of these enzymes, although posttranslational inactivation of IDO may prevent metabolic steps that are subsequent and consequent to IDO; 3) overcoming the IDO-dependent blockade by provision of a downstream quinolinate precursor activates the pathway and leads to the onset of suppressive properties; and 4) tolerogenic DCs can confer suppressive ability on otherwise immunogenic DCs across a Transwell in an IDO-dependent fashion. Altogether, these data indicate that kynurenine pathway enzymes downstream of IDO can initiate tolerogenesis by DCs independently of tryptophan deprivation. The paracrine production of kynurenines might be one mechanism used by IDO-competent cells to convert DCs lacking functional IDO to a tolerogenic phenotype within an IFN-gamma-rich environment.

The novel mechanism of peripheral tolerance

The circulating pool of lymphocytes contains self- as well as non-self reactive T cells. The mature DCs present as non-self as well as self-epitopes to naı̈ve T cells. To avoid autoimmunity the organism has to keep the mature DCs afar from self-specific T cells. I had proposed that the different anatomical distribution of the immature (tolerogenic) and mature (immunogenic) DCs in the peripheral lymphoid tissues may contribute to this mechanism. I had proposed that T cells to reach the mature DC should pass through the layer of immature DCs, which constitutively phagocytose and transport apoptotic cells to the regional LN and present only ‘self’ epitopes. Thus, self-specific T cells will be trapped by the immature DC layer and be deleted or become anergic. The layer of immature DC will be crossed only by those T cells that are not self-specific.