Immunogenic Dendritic Cells Research Articles

Induction of antigen-specific immune tolerance using biodegradable nanoparticles containing antigen and dexamethasone.

PurposeDexamethasone (Dex) has long been used as a potent immunosuppressive agent in the treatment of inflammatory and autoimmune diseases, despite serious side effects. In the present study, Dex and model antigen ovalbumin (OVA) were encapsulated with poly(lactic-co-glycolic acid) to deliver Dex and OVA preferentially to phagocytic cells, reducing systemic side effects of Dex. The OVA-specific immune tolerance-inducing activity of the nanoparticles (NPs) was examined.MethodsPolymeric NPs containing OVA and Dex (NP[OVA+Dex]) were prepared by the water-in-oil-in-water double emulsion solvent evaporation method. The effects of NP[OVA+Dex] on the maturation and function of immature dendritic cells (DCs) were examined in vitro. Furthermore, the OVA-specific immune tolerizing effects of NP[OVA+Dex] were confirmed in mice that were intravenously injected or orally fed with the NPs.ResultsImmature DCs treated in vitro with NP[OVA+Dex] did not mature into immunogenic DCs but instead were converted into tolerogenic DCs. Furthermore, profoundly suppressed generation of OVA-specific cytotoxic T cells and production of OVA-specific IgG were observed in mice injected with NP[OVA+Dex], whereas regulatory T cells were concomitantly increased. Feeding of mice with NP[OVA+Dex] also induced OVA-specific immune tolerance.ConclusionThe present study demonstrates that oral feeding as well as intravenous injection of poly(lactic-co-glycolic acid) NPs encapsulating both antigen and Dex is a useful means of inducing antigen-specific immune tolerance, which is crucial for the treatment of autoimmune diseases.

The Cross-Talk between miR-511-3p and C-Type Lectin Receptors on Dendritic Cells Affects Dendritic Cell Function.

MicroRNAs are small, noncoding RNAs that function as posttranscriptional modulators of gene expression by binding target mRNAs and inhibiting translation. They are therefore crucial regulators of several biological as well as immunological events. Recently, miR-511-3p has been implicated in the development and differentiation of APCs, such as dendritic cells (DCs), and regulating several human diseases. Interestingly, miR-511-3p is embedded within the human MRC1 gene that encodes the mannose receptor. In this study, we sought to examine the impact of miR-511-3p up- or downregulation on human DC surface phenotype, cytokine profile, immunogenicity (using IDO activity as a surrogate), and downstream T cell polarization. Using gene silencing and a selection of microRNA mimics, we could successfully suppress or induce the expression of miR-511-3p in DCs. Consequently, we show for the first time, to our knowledge, that inhibition and/or overexpression of miR-511-3p has opposing effects on the expression levels of two key C-type lectin receptors, namely the mannose receptor and DC-specific ICAM 3 nonintegrin at protein and mRNA levels, thereby affecting C-type lectin receptor-induced modulation of IDO activity in DCs. Furthermore, we show that downregulation of miR-511-3p drives an anti-inflammatory DC response characterized by IL-10 production. Interestingly, the miR-511-3plow DCs also promoted IL-4 secretion and suppressed IL-17 in cocultures with autologous T cells. Together, our data highlight the potential role of miR-511 in regulating DC function and downstream events leading to Th polarization and immune modulation.

STAT3 Activation in Combination with NF-KappaB Inhibition Induces Tolerogenic Dendritic Cells with High Therapeutic Potential to Attenuate Collagen-Induced Arthritis.

Dendritic cells (DCs) have the ability to induce tolerance or inflammation in response to self-antigens, which makes them fundamental players in autoimmunity. In this regard, immunogenic DCs produce IL-12 and IL-23 favouring the acquisition of Th1 and Th17 inflammatory phenotypes, respectively, by autoreactive CD4+ T-cells, thus promoting autoimmunity. Conversely, tolerogenic DCs produce IL-10 and TGF-β, inducing the generation of CD4+ T-cells with suppressive activity (Treg), which promote tolerance to self-constituents. Previous studies have shown that STAT3 signalling in DCs attenuates the production of proinflammatory cytokines, whilst NF-κB activation promotes it. In this study, we aimed to generate DCs displaying strong and constitutive tolerogenic profile to be used as immunotherapy in autoimmunity. To this end, we transduced bone marrow-derived DCs with lentiviral particles codifying for a constitutively active version of STAT3 (constitutively active STAT3 (STAT3ca)) or with a constitutive repressor of NF-κB (IκBα superrepressor (IκBαSR)), and their therapeutic potential was evaluated in a mouse model of arthritis induced by collagen (CIA). Our results show that STAT3ca transduction favoured the production of the anti-inflammatory mediator IL-10, whereas IκBαSR transduction attenuated the expression of the proinflammatory cytokine IL-23 in DCs. Moreover, both STAT3ca-transduced and IκBαSR-transduced DCs separately exerted a mild but significant therapeutic effect reducing the severity of CIA development. Furthermore, when DCs were transduced with both STAT3ca and IκBαSR together, they reduced CIA manifestation significantly stronger than when transduced with only STAT3ca or IκBαSR separately. These results show STAT3 and NF-κB as two important and complementary regulators of the tolerogenic behaviour of DCs, which should be considered as molecular targets in the design of DC-based suppressive immunotherapies for the treatment of autoimmune disorders.

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP's clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

Diacylglycerol Lipase-β Is Required for TNF-α Response but Not CD8+ T Cell Priming Capacity of Dendritic Cells

Diacylglycerol lipase-β (DAGLβ) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLβ regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive Tcell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLβ in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor α secretion. Cell-based vaccination studies revealed that DC maturation exvivo and immunogenicity invivo was surprisingly unaffected by DAGLβ inactivation. Collectively, we identify DAGLβ pathways as a means for attenuatingDC inflammatory signaling while sparing criticaladaptive immune functions and further expand theutility of targeting lipid pathways for immunomodulation.

Metabolic Control of Dendritic Cell Functions: Digesting Information.

Dendritic cells (DCs) control innate and adaptive immunity by patrolling tissues to gather antigens and danger signals derived from microbes and tissue. Subsequently, DCs integrate those environmental cues, orchestrate immunity or tolerance, and regulate tissue homeostasis. Recent advances in the field of immunometabolism highlight the notion that immune cells markedly alter cellular metabolic pathways during differentiation or upon activation, which has important implications on their functionality. Previous studies showed that active oxidative phosphorylation in mitochondria is associated with immature or tolerogenic DCs, while increased glycolysis upon pathogen sensing can promote immunogenic DC functions. However, new results in the last years suggest that regulation of DC metabolism in steady state, after immunogenic activation and during tolerance in different pathophysiological settings, may be more complex. Moreover, ontogenically distinct DC subsets show different functional specializations to control T cell responses. It is, thus, relevant how metabolism influences DC differentiation and plasticity, and what potential metabolic differences exist among DC subsets. Better understanding of the emerging connection between metabolic adaptions and functional DC specification will likely allow the development of therapeutic strategies to manipulate immune responses.

Cells to Surgery Quiz: May 2019

Cells to Surgery Quiz: May 2019

LKB1 expressed in dendritic cells governs the development and expansion of thymus-derived regulatory T cells.

Liver Kinase B1 (LKB1) plays a key role in cellular metabolism by controlling AMPK activation. However, its function in dendritic cell (DC) biology has not been addressed. Here, we find that LKB1 functions as a critical brake on DC immunogenicity, and when lost, leads to reduced mitochondrial fitness and increased maturation, migration, and T cell priming of peripheral DCs. Concurrently, loss of LKB1 in DCs enhances their capacity to promote output of regulatory T cells (Tregs) from the thymus, which dominates the outcome of peripheral immune responses, as suggested by increased resistance to asthma and higher susceptibility to cancer in CD11cΔLKB1 mice. Mechanistically, we find that loss of LKB1 specifically primes thymic CD11b+ DCs to facilitate thymic Treg development and expansion, which is independent from AMPK signalling, but dependent on mTOR and enhanced phospholipase C β1-driven CD86 expression. Together, our results identify LKB1 as a critical regulator of DC-driven effector T cell and Treg responses both in the periphery and the thymus.

Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells.

Dendritic cells (DCs) are pivotal in regulating tolerogenic as well as immunogenic responses against microorganisms by directing both the innate and adaptive immune response. In health, phenotypically different DC subsets found in the gut mucosa are maintained in their tolerogenic state but switch to a pro-inflammatory phenotype during infection or chronic autoinflammatory conditions such as inflammatory bowel disease (IBD). The mechanisms that promote the switch among the mucosal DCs from a tolerogenic to an immunogenic, pro-inflammatory phenotype are incompletely understood. We hypothesized that disabled homolog 2 (DAB2), recently described as a negative regulator of DC immunogenicity during their development, is regulated during intestinal inflammation and modulates mucosal DC function. We show that DAB2 is highly expressed in colonic CD11b+CD103− DCs, a subset known for its capacity to induce inflammatory Th1/Th17 responses in the colon, and is downregulated predominantly in this DC subset during adoptive T cell transfer colitis. Administration of Dab2-deficient DCs (DC2.4Dab2−/− cells) modulated the course of DSS colitis in wild-type mice, enhanced mucosal expression of Tnfa, Il6, and Il17a, and promoted neutrophil recruitment. In bone-marrow derived dendritic cells (BMDC), DAB2 expression correlated with CD11b levels and DAB2 was rapidly and profoundly inhibited by TLR ligands in a TRIF- and MyD88-dependent manner. The negative modulation of DAB2 was biphasic, initiated with a quick drop in DAB2 protein, followed by a sustained reduction in Dab2 mRNA. DAB2 downregulation promoted a more functional and activated DC phenotype, reduced phagocytosis, and increased CD40 expression after TLR activation. Furthermore, Dab2 knockout in DCs inhibited autophagy and promoted apoptotic cell death. Collectively, our results highlight the immunoregulatory role for DAB2 in the intestinal dendritic cells and suggest that DAB2 downregulation after microbial exposure promotes their switch to an inflammatory phenotype.

Are Conventional Type 1 Dendritic Cells Critical for Protective Antitumor Immunity and How?

Dendritic cells (DCs) are endowed with a unique potency to prime T cells, as well as to orchestrate their expansion, functional polarization and effector activity in non-lymphoid tissues or in their draining lymph nodes. The concept of harnessing DC immunogenicity to induce protective responses in cancer patients was put forward about 25 years ago and has led to a multitude of DC-based vaccine trials. However, until very recently, objective clinical responses were below expectations. Conventional type 1 DCs (cDC1) excel in the activation of cytotoxic lymphocytes including CD8+ T cells (CTLs), natural killer (NK) cells, and NKT cells, which are all critical effector cell types in antitumor immunity. Efforts to investigate whether cDC1 might orchestrate immune defenses against cancer are ongoing, thanks to the recent blossoming of tools allowing their manipulation in vivo. Here we are reporting on these studies. We discuss the mouse models used to genetically deplete or manipulate cDC1, and their main caveats. We present current knowledge on the role of cDC1 in the spontaneous immune rejection of tumors engrafted in syngeneic mouse recipients, as a surrogate model to cancer immunosurveillance, and how this process is promoted by type I interferon (IFN-I) effects on cDC1. We also discuss cDC1 implication in promoting the protective effects of immunotherapies in mouse preclinical models, especially for adoptive cell transfer (ACT) and immune checkpoint blockers (ICB). We elaborate on how to improve this process by in vivo reprogramming of certain cDC1 functions with off-the-shelf compounds. We also summarize and discuss basic research and clinical data supporting the hypothesis that the protective antitumor functions of cDC1 inferred from mouse preclinical models are conserved in humans. This analysis supports potential applicability to cancer patients of the cDC1-targeting adjuvant immunotherapies showing promising results in mouse models. Nonetheless, further investigations on cDC1 and their implications in anti-cancer mechanisms are needed to determine whether they are the missing key that will ultimately help switching cold tumors into therapeutically responsive hot tumors, and how precisely they mediate their protective effects.

IL-15 and a Two-Step Maturation Process Improve Bone Marrow-Derived Dendritic Cell Cancer Vaccine.

In the last 20 years, dendritic cells (DCs) have been largely used as a platform for therapeutic vaccination in cancer patients. However, despite its proven safety and ability to induce cancer specific immune responses, the clinical benefits of DC-based immunotherapy are currently very limited. Thus, novel approaches are still needed to boost its efficacy. Our group recently showed that squaric acid treatment of antigens is an important adjuvant that can increase vaccine-induced downstream immune responses and therapeutic outcomes. Here we further improved this dendritic cell vaccine formulation by developing a new method for differentiating and maturing DCs from their bone marrow precursors. Our data demonstrate that bone marrow-derived DCs differentiated with GM-CSF and IL-15 and matured with a maturation cocktail in two steps present a more mature and immunogenic phenotype, compared to standard DC preparations. Further suppression of the prostaglandin E2 pathway achieved even more immunogenic DC phenotypes. This vaccine was more potent at delaying tumor growth, improved animal survival and induced a more immunogenic and Th1-skewed T cell response in an ovarian cancer mouse model. These promising results support future efforts for the clinical translation of this approach.

Polymeric Nanoparticles Containing Both Antigen and Vitamin D3 Induce Antigen-Specific Immune Suppression.

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (aVD3), is known to exert beneficial effects in the treatment of autoimmune diseases because of its immunosuppressive effects. However, clinical application of aVD3 remains limited because of the potential side effects, particularly hypercalcemia. Encapsulation of aVD3 within biodegradable nanoparticles (NPs) would enhance the delivery of aVD3 to antigen presenting cells, while preventing the potential systemic side effects of aVD3. In the present study, polymeric NPs containing ovalbumin (OVA) and aVD3 (NP[OVA+aVD3]) were prepared via the water-in-oil-in-water double emulsion solvent evaporation method, after which their immunomodulatory effects were examined. Bone marrow-derived immature dendritic cells (DCs) treated with NP(OVA+aVD3) did not mature into immunogenic DCs but were converted into tolerogenic DCs, which express low levels of co-stimulatory molecules and MHC class II molecules, produce lower levels of pro-inflammatory cytokines while increasing the production of IL-10 and TGF-β, and induce the generation of Tregs. Intravenous injection with NP(OVA+aVD3) markedly suppressed the generation of OVA-specific CTLs in mice. Furthermore, OVA-specific immune tolerance was induced in mice orally administered with NP(OVA+aVD3). These results show that biodegradable NPs encapsulating both antigen and aVD3 can effectively induce antigen-specific immune suppression.

Role of the p38 MAPK/C/EBPβ Pathway in the Regulation of Phenotype and IL-10 and IL-12 Production by Tolerogenic Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) play a major role in innate and adaptive immunity and self-immune tolerance. Immunogenic versus tolerogenic DC functions are dictated by their levels of costimulatory molecules and their cytokine expression profile. The transcription factor C/EBPβ regulates the expression of several inflammatory genes in many cell types including macrophages. However, little is known regarding the role of C/EBPβ in tolerogenic versus immunogenic DCs functions. We have previously reported that bone marrow-derived DCs generated with GM-CSF (GM/DCs) acquire the signature of semi-mature tolerogenic IL-10-producing DCs as opposed to immunogenic DCs generated with GM-CSF and IL-4 (IL-4/DCs). Here, we show that tolerogenic GM/DCs exhibit higher levels of phosphorylation and enhanced DNA binding activity of C/EBPβ and CREB than immunogenic IL-4/DCs. We also show that the p38 MAPK/CREB axis and GSK3 play an important role in regulating C/EBPβ phosphorylation and DNA binding activity. Inhibition of p38 MAPK in GM/DCs resulted in a drastic decrease of C/EBPβ and CREB DNA binding activities, a reduction of their IL-10 production and an increase of their IL-12p70 production, a characteristic of immunogenic IL-4/DCs. We also present evidence that GSK3 inhibition in GM/DCs reduced C/EBPβ DNA binding activity and increased expression of costimulatory molecules in GM/DCs and their production of IL-10. Analysis of GM/DCs of C/EBPβ−/− mice showed that C/EBPβ was essential to maintain the semimature phenotype and the production of IL-10 as well as low CD4+ T cell proliferation. Our results highlight the importance of the p38MAPK-C/EBPβ pathway in regulating phenotype and function of tolerogenic GM/DCs.

Tumor-Derived Microvesicles Enhance Cross-Processing Ability of Clinical Grade Dendritic Cells.

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighboring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens, and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccines.

Antigen-loaded dendritic cells triggers a specific cytotoxic Tlymphocytes immune response against hepatocellular carcinoma: in vitro study.

Hepatocellular carcinoma (HCC) is one of the common malignancies, characterized by poor response to conventional therapeutic options. Immunotherapy with dendritic cells (DCs)-vaccines is one of the most successful strategies used for the treatment of HCC. However, the methods applied in the preparation of antigen-loaded DCs are important factors for optimization of DCs vaccines. The present study was conducted to investigate the effect of HCC-whole tumor cell lysate prepared using rapid repetitive freeze-thaw cycles on the immunogenicity of DCs and evaluate the ability of whole tumor cell lysate-pulsed DCs vaccine to induce a specific cytotoxic Tlymphocytes (CTLs) response against HepG2 cell line. Immature DCs generated from peripheral blood monocytes were randomized into two groups: control DCs and whole tumor cell lysate-pulsed DCs. Phenotypic analysis of the DCs' cell maturation marker CD83 and co-stimulatory molecule CD86 was performed. HCC-specific cytotoxic activity of CD8+ CTLs was measured in vitro. Loading of DCs with necrotic whole cell lysate resulted in non-significant changes in DCs' expression of CD83, but a significant increase in expression of CD86. In addition, CD8+ CTLs stimulated with whole tumor cell lysate-pulsed DCs showed a high cytotoxic activity that specifically attack HepG2 cells. Our findings indicated that pulsation of DCs with whole tumor cell lysate prepared by repetitive freeze-thaw cycles could efficiently enhance the ability of DCs to induce proliferation and clonal expansion of CD8+ CTLs. Data herein, also indicated that whole tumor cell lysate-pulsed DCs triggers a specific CD8+ CTLs against HCC tumor cells.

Synergistic Antimyeloma Activity of Dendritic Cells and Pomalidomide in a Murine Myeloma Model.

We have previously shown that immunization with tumor antigen-loaded dendritic cells (DCs) and the immunomodulating drug, lenalidomide, synergistically potentiates the enhancing antitumor immunity in a myeloma mouse model. In this study, we investigated the immunogenicity of DCs combined with pomalidomide and dexamethasone in a myeloma mouse model. MOPC-315 cells were injected subcutaneously to establish myeloma-bearing mice. Four test groups were used to mimic clinical protocol: (1) PBS control, (2) DCs, (3) pomalidomide + dexamethasone, and (4) DCs + pomalidomide + dexamethasone. The combination of DCs plus pomalidomide and dexamethasone displayed greater inhibition of tumor growth compared to the other groups. This effect was closely related with reduced numbers of immune suppressor cells including myeloid-derived suppressor cells, M2 macrophages, and regulatory T cells, with the induction of immune effector cells such as CD4+ and CD8+ T cells, memory T cells, natural killer (NK) cells, and M1 macrophages, and with the activation of T lymphocytes and NK cells in the spleen. Moreover, the level of the immunosuppressive factor vascular endothelial growth factor was significantly reduced in the tumor microenvironment. The collective findings in the murine myeloma model suggest that tumor antigen-loaded DCs combined with pomalidomide and dexamethasone synergistically enhance antitumor immunity by skewing the immune-suppressive status toward an immune-supportive status.

TNP-470 skews DC differentiation to Th1-stimulatory phenotypes and can serve as a novel adjuvant in a cancer vaccine

AbstractFumagillin is an antiangiogenic and antineoplastic fungal natural product, and TNP-470 is one of its most potent analogs. Decades of studies revealed that TNP-470 has potent anticancer activities via destruction of neovasculature. In stark contrast, TNP-470 has been reported to suppress lymphocyte proliferation, thereby limiting its clinical potentials. In an attempt to investigate whether the similar or opposite immunomodulatory effect of TNP-470 could act on myeloid cells, we found that TNP-470 potentiates the immunogenicity of dendritic cells (DCs) toward a phenotype with T helper cell type 1 (Th1)–stimulatory features. Using DC vaccine on a murine melanoma cancer model, the TNP-470–treated DC vaccine could significantly induce tumor-specific immunogenicity and substantially enhance tumor eradication when compared with vehicle-treated DC vaccine in a prophylactic setting. Enhanced tumor-specific immunogenicity and delayed tumor progression were observed in a therapeutic setting upon the TNP-470–treated DC vaccine. Our data showed that TNP-470 potentiates Toll-like receptor signaling, including NF-κB activation, in DCs to transcriptionally activate interleukin-12 production, thus inducing a Th1-immune response. Our current study uncovers a novel immune function of TNP-470 in DCs and redefines its role as a novel class of small molecule immune adjuvant in DC-based cancer vaccine given potentiation of DC immunogenicity is a major roadblock in DC vaccine development. Our study not only provides a novel adjuvant for ex vivo–cultured patient-specific DC vaccines for cancer treatment but also discovers the distinct immunostimulatory function of TNP-470 in DCs of myeloid lineage that differs from its immunosuppressive function in lymphoid cells.

Tumor lysate-loaded Bacterial Ghosts as a tool for optimized production of therapeutic dendritic cell-based cancer vaccines

Cancer immunotherapy with dendritic cell (DC)-based vaccines has been used to treat various malignancies for more than two decades, however generally showed a limited clinical success. Among various factors responsible for their modest clinical activity is the lack of universally applied, standardized protocols for the generation of clinical-grade DC vaccines, capable of inducing effective anti-tumor immune responses. We investigated Bacterial Ghosts (BGs) – empty envelopes of Gram-negative bacteria – as a tool for optimized production of DC vaccines. BGs possess various intact cell surface structures, exhibiting strong adjuvant properties required for the induction of DC maturation, whereas their empty internal space can be easily filled with a source tumor antigens, e.g. tumor lysate. Hence BGs emerge as an excellent platform for both the induction of immunogenic DC maturation and loading with tumor antigens in a single-step procedure. We compared the phenotype, cytokine secretion profile, functional activity and ability to induce immunogenic T-cell responses in vitro of human monocyte-derived DCs generated using BG platform and DCs matured with widely used lipopolysaccharide (LPS) plus interferon-γ cocktail and loaded with tumor lysate. Both approaches induced DC maturation, however BG-based protocol was superior to LPS-based protocol in terms of the ability to induce DCs with a lower tolerogenic potential, resulting in a more robust CD8+ T cell activation and their functional activity as well as significantly lower induction of regulatory T cells. These superior parameters are attributed, at least in part, to the ability of BG-matured DCs to resist potential immunosuppressive and pro-tolerogenic activity of various tumor cell lysates, including melanoma, renal carcinoma and glioblastoma.

Blockade of MIF-CD74 Signalling on Macrophages and Dendritic Cells Restores the Antitumour Immune Response Against Metastatic Melanoma.

Mounting an effective immune response against cancer requires the activation of innate and adaptive immune cells. Metastatic melanoma is the most aggressive form of skin cancer. While immunotherapies have shown a remarkable success in melanoma treatment, patients develop resistance by mechanisms that include the establishment of an immune suppressive tumor microenvironment. Thus, understanding how metastatic melanoma cells suppress the immune system is vital to develop effective immunotherapies against this disease. In this study, we find that macrophages (MOs) and dendritic cells (DCs) are suppressed in metastatic melanoma and that the Ig-CDR-based peptide C36L1 is able to restore MOs and DCs’ antitumorigenic and immunogenic functions and to inhibit metastatic growth in lungs. Specifically, C36L1 treatment is able to repolarize M2-like immunosuppressive MOs into M1-like antitumorigenic MOs, and increase the number of immunogenic DCs, and activated cytotoxic T cells, while reducing the number of regulatory T cells and monocytic myeloid-derived suppressor cells in metastatic lungs. Mechanistically, we find that C36L1 directly binds to the MIF receptor CD74 which is expressed on MOs and DCs, disturbing CD74 structural dynamics and inhibiting MIF signaling on these cells. Interfering with MIF–CD74 signaling on MOs and DCs leads to a decrease in the expression of immunosuppressive factors from MOs and an increase in the capacity of DCs to activate cytotoxic T cells. Our findings suggest that interfering with MIF–CD74 immunosuppressive signaling in MOs and DCs, using peptide-based immunotherapy can restore the antitumor immune response in metastatic melanoma. Our study provides the rationale for further development of peptide-based therapies to restore the antitumor immune response in metastatic melanoma.

Lycium barbarum Polysaccharide Promotes Maturation of Dendritic Cell via Notch Signaling and Strengthens Dendritic Cell Mediated T Lymphocyte Cytotoxicity on Colon Cancer Cell CT26-WT.

Lycium barbarum polysaccharide (LBP) is the major function component of Lycium barbarum L. and has been previously reported to induce the phenotypic and functional maturation of dendritic cells (DCs) as well as activating T lymphocytes. In the current study, the immunologic cytotoxicity promoting effect of LBP was assessed and the underlying mechanism was explored. The impact of LBP on the phenotype, maturation, and immunogenicity of DCs was assessed. The activity of Notch pathway which is involved in the regulation of LBP on DCs was detected. Afterwards, the influence of LBP on cytotoxicity of DC-mediated cytotoxicity T lymphocytes (CTLs) to CT26-WT colon cancer cells was further assessed. Administration of LBP induced the phenotypic and functional maturation of DCs. After being subjected to LBP, the expression of Notch and Jagged and Notch targets Hes1 and Hes5 was all upregulated. The cytotoxicity of DC-mediated CTLs was strengthened by administration of LBP. Additionally, cytotoxicity of DC-mediated CTLs on CT26-WT colon cancer cells also increased with effector-target ratio. In conclusion, LBP could induce the phenotypic and functional maturation of DCs via Notch signaling and promote the cytotoxicity of DC-mediated CTLs, which could be employed as a promising adjuvant for cancer immunotherapy.