Properties Of Dendritic Cells Research Articles

Injectable Micro-Hydrogel for DNA Delivery: A Promising Therapeutic Platform.

Utilizing the immune system as a strategy for disease prevention and treatment is promising, especially with dendritic cells (DCs) playing a central role in adaptive immune responses. The unique properties of DCs drive interest in developing materials for cell-based therapy and immune modulation. Injectable systems require syringe-compatible scaffolds, while hydrogels, like alginate, known for their programmability and biocompatibility, offer a versatile platform for immune medicine enhancement through easy preparation and room-temperature cross-linking. In this study, we synthesized alginate balls loaded with DCs or cytosine-phosphorothioate-guanine deoxyribonucleotide (CpG DNA) microparticles, aiming for long-term immune cell culture with potential immune stimulation effects. Encapsulated DCs exhibited proliferation within the alginate balls for up to 7 days, and CpG MPs were uniformly dispersed, which can facilitate uptake by DCs. This was supported by the result that DCs effectively phagocytosed CpG microparticles in a 2D environment. After the uptake of CpG MPs, the alginate balls with CpG-MP-uptaken DCs were synthesized successfully. The injectable properties of the alginate balls were easily modulated by adjusting the syringe needle gauges. This innovative strategy holds substantial promise for advancing medical treatments, offering effective and comfortable solutions for controlled immune modulation.

Mechanism of TLR4-Mediated Anti-Inflammatory Response Induced by Exopolysaccharide from the Probiotic Bacillus subtilis.

Intestinal inflammatory diseases affect millions of people worldwide, and one class of drugs showing promise toward treatment of several inflammatory diseases is probiotics. Numerous studies have been performed using probiotics to prevent and treat intestinal inflammatory diseases. Most of these studies used intact bacteria, and neither the active molecule nor the molecular mechanisms by which they affect immune responses are known. We have shown that the probiotic Bacillus subtilis is anti-inflammatory and can protect mice from acute colitis induced by the enteric pathogen Citrobacter rodentium. We identified and purified the active molecule, exopolysaccharide (EPS), and showed that it protects mice from C. rodentium-induced colitis by inducing anti-inflammatory M2 macrophages or inhibitory dendritic cells (DCs), both of which inhibit excessive T cell responses. We showed previously that EPS affects macrophages and DCs in a TLR4-dependent manner, and in the current study we asked how EPS induces these anti-inflammatory cells and how they function to inhibit T cells. By investigating the signaling downstream of TLR4 that leads to acquisition of inhibitory properties of macrophages and DCs, we found that EPS induces expression of the inhibitory molecule IDO in bone marrow-derived DCs, and that inhibition of T cell proliferation by IDO-expressing bone marrow-derived DCs utilizes the kynurenine/aryl hydrocarbon receptor circuit. Furthermore, unlike LPS, EPS does not induce inflammatory cytokines upon injection invivo, directly demonstrating different outcomes induced by two different TLR4 agonists.

The Epigenetic Reader Protein SP140 Regulates Dendritic Cell Activation, Maturation and Tolerogenic Potential

SP140 is an epigenetic reader protein expressed predominantly in immune cells. GWAS studies have shown an association between SP140 single nucleotide polymorphisms (SNPs) and diverse autoimmune and inflammatory diseases, suggesting a possible pathogenic role for SP140 in immune-mediated diseases. We previously demonstrated that treatment of human macrophages with the novel selective inhibitor of the SP140 protein (GSK761) reduced the expression of endotoxin-induced cytokines, implicating a role of SP140 in the function of inflammatory macrophages. In this study, we investigated the effects of GSK761 on in vitro human dendritic cell (DC) differentiation and maturation, assessing the expression of cytokines and co-stimulatory molecules and their capacity to stimulate T-cell activation and induce phenotypic changes. In DCs, lipopolysaccharide (LPS) stimulation induced an increase in SP140 expression and its recruitment to transcription start sites (TSS) of pro-inflammatory cytokine genes. Moreover, LPS-induced cytokines such as TNF, IL-6, and IL-1β were reduced in GSK761- or SP140 siRNA- treated DCs. Although GSK761 did not significantly affect the expression of surface markers that define the differentiation of CD14+ monocytes into immature DCs (iDCs), subsequent maturation of iDCs to mature DCs was significantly inhibited. GSK761 strongly reduced expression of the maturation marker CD83, the co-stimulatory molecules CD80 and CD86, and the lipid-antigen presentation molecule CD1b. Finally, when the ability of DCs to stimulate recall T-cell responses by vaccine-specific T cells was assessed, T cells stimulated by GSK761-treated DCs showed reduced TBX21 and RORA expression and increased FOXP3 expression, indicating a preferential generation of regulatory T cells. Overall, this study suggests that SP140 inhibition enhances the tolerogenic properties of DCs, supporting the rationale of targeting SP140 in autoimmune and inflammatory diseases where DC-mediated inflammatory responses contribute to disease pathogenesis.

High extracellular sodium chloride concentrations induce resistance to LPS signal in human dendritic cells.

Recent evidence showed that in response to elevated sodium dietary intakes, many body tissues retain Na+ ions for long periods of time and can reach concentrations up to 200mM. This could modulate the immune system and be responsible for several diseases. However, studies brought contrasted results and the effects of external sodium on human dendritic cell (DC) responses to danger signals remain largely unknown. Considering their central role in triggering T cell response, we tested how NaCl-enriched medium influences human DCs properties. We found that DCs submitted to high extracellular Na+ concentrations up to 200mM remain viable and maintain the expression of specific DC markers, however, their maturation, chemotaxis toward CCL19, production of pro-inflammatory cytokines and ROS in response to LPS were also partially inhibited. In line with these results, the T-cell allostimulatory capacity of DCs was also inhibited. Finally, our data indicate that high NaCl concentrations triggered the phosphorylation of SGK1 and ERK1/2 kinases. These results raised the possibility that the previously reported pro-inflammatory effects of high NaCl concentrations on T cells might be counterbalanced by a downregulation of DC activation.

Lung Tumor Cells with Different Tn Antigen Expression Present Distinctive Immunomodulatory Properties.

Lung cancer is the first leading cause of cancer-related deaths in the world. Aberrant glycosylation in lung tumors leads to the expression of tumor-associated carbohydrate structures, such as the Tn antigen, consisting of N-acetyl-galactosamine (GalNAc) linked to a serine or threonine residue in proteins (α-GalNAc-O-Ser/Thr). The Tn antigen can be recognized by the Macrophage Galactose/GalNAc lectin (MGL), which mediates various immune regulatory and tolerogenic functions, mainly by reprogramming the maturation of function of dendritic cells (DCs). In this work, we generated two different Tn-expressing variants from the Lewis-type lung murine cancer cell line LL/2, which showed different alterations in the O-glycosylation pathways that influenced the interaction with mouse MGL2 and the immunomodulatory properties of DCs. Thus, the identification of the biological programs triggered by Tn+ cancer cells might contribute to an improved understanding of the molecular mechanisms elicited by MGL-dependent immune regulatory circuits.

Triiodothyronine-stimulated dendritic cell vaccination boosts antitumor immunity against murine colon cancer

Given the ability of dendritic cells (DCs) to modulate other immune players, their manipulation holds great potential for inducing efficient antitumor immunity. However, DC vaccine manufacturing deserve optimization since tumor cell cargo and DC functional state induced by maturation signals influence their in vivo immunogenic potential. We reported that triiodothyronine (T3) stimulates mice DCs’ maturation and their ability to promote pro-inflammatory and cytotoxic T-cell responses. This study aimed to evaluate the efficacy of T3-conditioned DC vaccination in a murine model of colon carcinoma, deciphering the molecular players involved; and to examine the effects of T3 on the maturation and activation of human DCs (huDCs). Bone marrow-derived DCs were exposed to T3 and MC38 cancer cells that underwent cell death (MC38-Apo/Nec) by UVB irradiation. Our results showed that MC38-Apo/Nec cells are efficiently uptaken by DCs and that T3 upregulates CD86 expression with increased production of the pro-inflammatory cytokines IL-12, IL-6 and TGF-β. In a colon cancer model, vaccination with T3-stimulated and tumor antigen-loaded DCs inhibited tumor growth in wild type mice, an effect that was eliminated in IL-17-deficient animals. Notably, secretion of high levels of IFN-γ and IL-17, induction of Th1, Th17 and tumor-specific cytotoxic T lymphocytes characterized the antitumor response upon vaccination. Moreover, our initial findings demonstrated a significant increase in CD86 expression and IL-12 production by huDCs induced by T3. Overall, these results reinforce the adjuvant properties of T3-conditioned DCs to potentiate T-cell-mediated antitumor immunity and support promising advances in the translation process to human oncotherapy.

CTLA-4 silencing in dendritic cells loaded with colorectal cancer cell lysate improves autologous T cell responses in vitro.

Dendritic cell (DC)-based immunotherapy has increased interest among anti-cancer immunotherapies. Nevertheless, the immunosuppressive mechanisms in the tumor milieu, e.g., inhibitory immune checkpoint molecules, have been implicated in diminishing the efficacy of DC-mediated anti-tumoral immune responses. Therefore, the main challenge is to overcome inhibitory immune checkpoint molecules and provoke efficient T-cell responses to antigens specifically expressed by cancerous cells. Among the inhibitory immune checkpoints, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression on DCs diminishes their maturation and antigen presentation capability. Accordingly, we hypothesized that the expression of CTLA-4 on DCs inhibits the T cell-mediated anti-tumoral responses generated following the presentation of tumor antigens by DCs to T lymphocytes. In this study, we loaded colorectal cancer (CRC) cell lysate on DCs and inhibited the expression of CTLA-4 by small interfering RNA (siRNA) in them to investigate the DCs’ functional and phenotypical features, and T-cell mediated responses following DC/T cell co-culture. Our results demonstrated that blockade of CTLA-4 could promote stimulatory properties of DCs. In addition, CTLA-4 silenced CRC cell lysate-loaded DCs compared to the DCs without CTLA-4 silencing resulted in augmented T cell proliferation and cytokine production, i.e., IFN-γ and IL-4. Taken together, our findings suggest CTLA-4 silenced CRC cell lysate-loaded DCs as a promising therapeutic approach however further studies are needed before this strategy can be used in clinical practice.

The PDE4 inhibitor tanimilast shows distinct immunomodulatory properties associated with a type 2 endotype and CD141 upregulation

BackgroundTanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast.MethodsDCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarization. The regulation of type 2-skewing mediators was investigated by real-time PCR in DCs and compared to results obtained in vivo in a randomized placebo-controlled trial on COPD patients treated with tanimilast.ResultsOur results show that both tanimilast and budesonide reduced the production of the immunostimulatory cytokine IFN-γ by CD4+ T cells. However, the two drugs acted at different levels since budesonide mainly blocked T cell proliferation, while tanimilast skewed T cells towards a Th2 phenotype without affecting cell proliferation. In addition, only DCs matured in the presence of tanimilast displayed increased CD86/CD80 ratio and CD141 expression, which correlated with Th2 T cell induction and dead cell uptake respectively. These cells also upregulated cAMP-dependent immunosuppressive molecules such as IDO1, TSP1, VEGF-A and Amphiregulin. Notably, the translational value of these data was confirmed by the finding that these same genes were upregulated also in sputum cells of COPD patients treated with tanimilast as add-on to inhaled glucocorticoids and bronchodilators.ConclusionTaken together, these findings demonstrate distinct immunomodulatory properties of tanimilast associated with a type 2 endotype and CD141 upregulation in DCs and provide a mechanistic rationale for the administration of tanimilast on top of inhaled corticosteroids.

129P Regulatory dendritic cells correlate with an altered T cell distribution and regulatory T cell phenotype in metastatic lymph nodes of NSCLC patients

Dendritic cells (DCs) are considered to be the most important professional population of antigen-presenting cells and play a crucial role in the activation of the immune system. However, the proper function of DCs is altered by the tumor microenvironment (TME). Despite the progress in anticancer immunotherapy, there are still many unresolved issues. Among them, the immunomodulatory properties of DCs in lung cancer are of interest. The aim of the study was to analyze the PD-1+ PD-L1+ CD80- DCs subset, described as regulatory dendritic cells (DCregs), and to investigate correlations between the presence of DCregs and changes in T cell frequencies and immunomodulatory molecules in metastatic and non-metastatic lymph nodes (LNs) aspirates of NSCLC patients. LNs aspirates were obtained during the diagnostic EBUS TBNA procedure of 30 NSCLC patients. DCregs and T cell characteristics were determined by multiparameter flow cytometry. We noticed a higher percentage of DCregs in the metastatic than in the non-metastatic LNs (22.5% vs. 3.1%). DCs cells show increased expression of PD-1 and PD-L1. Additionally, metastatic LNs contained a lower percentage of CD4 T cells, whereas the percentage of CD8 T cells and Tregs was increased as compared to non-metastatic LNs. Surprisingly, the percentage of DCregs positively correlated with the percentage of PD-1+ T cells, Tregs and CD45RO+ Tregs. Our results confirm the utility of flow cytometric analysis of EBUS/TBNA samples to assess the interaction between immune cells in TME. DCregs strongly associates with an altered T cell distribution and immunosuppressive phenotype in metastatic LNs of NSCLC patients. It can be assumed that DCregs are involved in the suppression of anti-tumor response, which could be of importance for the response upon immunotherapy.

Research Highlights

Research Highlights

Immunosuppressive properties of human PD-1 + , PDL-1 + and CD80 + dendritic cells from lymph nodes aspirates of lung cancer patients.

Dendritic cells (DCs) play a pivotal role in the homeostasis of the immune system. The tumor microenvironment impairs the proper function of DCs. The immunomodulatory properties of DCs in lung cancer are of interest. In the present study, we analysed DCs subsets and immune cells with the expression of immunomodulatory molecules: PD-1 and PD-L1 and co-stimulatory molecule CD80 in metastatic, non-metastatic lymph nodes (LNs) and peripheral blood (PB). LNs aspirates were obtained during the EBUS/TBNA procedure of 29 patients with primary lung cancer. The cells were analyzed by flow cytometry. We reported a higher percentage of DCs in the metastatic than in the non-metastatic LNs and the PB (0.709% vs. 0.166% vs. 0.043%, p < 0.0001). The proportions of PD-1 + , PD-L1 + and CD80 + DCs were higher in the metastatic LNs than in the non-metastatic ones. A higher proportion of regulatory DCs (DCregs) was found in the metastatic ones than in the non-metastatic LNs (22.5% vs. 3.1%, p = 0.0189). We report that DCs cells show increased expression of PD-1, PD-L1 and CD80 molecules that can interact with T lymphocytes. It can be assumed that mature DCs infiltrating metastatic LNs can develop into DCregs, which are involved in the suppression of anti-tumor response.

Vitamin D receptor, STAT3, and TET2 cooperate to establish tolerogenesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, induces a stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis invitro. Differentiation to tolerogenic DCs associates with activation of the IL-6-JAK-STAT3 pathway. We show that JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of JAK2 reverts vitamin D-induced tolerogenic properties of DCs. This interplay among VDR, STAT3, and TET2 opens up possibilities for modulating DC immunogenic properties in clinics.

How dendritic cells sense and respond to viral infections.

The ability of dendritic cells (DCs) to sense viral pathogens and orchestrate a proper immune response makes them one of the key players in antiviral immunity. Different DC subsets have complementing functions during viral infections, some specialize in antigen presentation and cross-presentation and others in the production of cytokines with antiviral activity, such as type I interferons. In this review, we summarize the latest updates concerning the role of DCs in viral infections, with particular focus on the complex interplay between DC subsets and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Despite being initiated by a vast array of immune receptors, DC-mediated antiviral responses often converge towards the same endpoint, that is the production of proinflammatory cytokines and the activation of an adaptive immune response. Nonetheless, the inherent migratory properties of DCs make them a double-edged sword and often viral recognition by DCs results in further viral dissemination. Here we illustrate these various aspects of the antiviral functions of DCs and also provide a brief overview of novel antiviral vaccination strategies based on DCs targeting.

Cannabinoids induce functional Tregs by promoting tolerogenic DCs via autophagy and metabolic reprograming

The generation of functional regulatory T cells (Tregs) is essential to keep tissue homeostasis and restore healthy immune responses in many biological and inflammatory contexts. Cannabinoids have been pointed out as potential therapeutic tools for several diseases. Dendritic cells (DCs) express the endocannabinoid system, including the cannabinoid receptors CB1 and CB2. However, how cannabinoids might regulate functional properties of DCs is not completely understood. We uncover that the triggering of cannabinoid receptors promote human tolerogenic DCs that are able to prime functional FOXP3+ Tregs in the context of different inflammatory diseases. Mechanistically, cannabinoids imprint tolerogenicity in human DCs by inhibiting NF-κB, MAPK and mTOR signalling pathways while inducing AMPK and functional autophagy flux via CB1- and PPARα-mediated activation, which drives metabolic rewiring towards increased mitochondrial activity and oxidative phosphorylation. Cannabinoids exhibit in vivo protective and anti-inflammatory effects in LPS-induced sepsis and also promote the generation of FOXP3+ Tregs. In addition, immediate anaphylactic reactions are decreased in peanut allergic mice and the generation of allergen-specific FOXP3+ Tregs are promoted, demonstrating that these immunomodulatory effects take place in both type 1- and type 2-mediated inflammatory diseases. Our findings might open new avenues for novel cannabinoid-based interventions in different inflammatory and immune-mediated diseases.

Vitamin D Receptor, STAT3 and TET2 Cooperate to Establish Tolerogenesis

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis. Tolerogenic properties of DCs are acquired together with activation of the IL6-JAK-STAT3 pathway. In fact, ChIP-seq analysis shows that VDR directly binds the IL6 gene, and JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of STAT3 phosphorylation reverts vitamin D-induced tolerogenic properties of DCs. This novel interplay between VDR, STAT3 and TET2 opens up possibilities for modulating DC immunogenic properties in the clinics.

Fecal microbiota composition associates with the capacity of human peripheral blood monocytes to differentiate into immunogenic dendritic cells in vitro

ABSTRACT Although promising for active immunization in cancer patients, dendritic cells (DCs) vaccines generated in vitro display high inter-individual variability in their immunogenicity, which mostly limits their therapeutic efficacy. Gut microbiota composition is a key emerging factor affecting individuals’ immune responses, but it is unknown how it affects the variability of donors’ precursor cells to differentiate into immunogenic DCs in vitro. By analyzing gut microbiota composition in 14 healthy donors, along with the phenotype and cytokines production by monocyte-derived DCs, we found significant correlations between immunogenic properties of DC and microbiota composition. Namely, donors who had higher α-diversity of gut microbiota and higher abundance of short-chain fatty acid (SCFAs) and SCFA-producing bacteria in feces, displayed lower expression of CD1a on immature (im)DC and higher expression of ILT-3, costimulatory molecules (CD86, CD40) proinflammatory cytokines (TNF-α, IL-6, IL-8) and IL-12p70/IL-10 ratio, all of which correlated with their lower maturation potential and immunogenicity upon stimulation with LPS/IFNγ, a well-known Th1 polarizing cocktail. In contrast, imDCs generated from donors with lower α-diversity and higher abundance of Bifidobacterium and Collinsella in feces displayed higher CD1a expression and higher potential to up-regulate CD86 and CD40, increase TNF-α, IL-6, IL-8 production, and IL-12p70/IL-10 ratio upon stimulation. These results emphasize the important role of gut microbiota on the capacity of donor precursor cells to differentiate into immunogenic DCs suitable for cancer therapy, which could be harnessed for improving the actual and future DC-based cancer therapies.

A WT-1 and PRAME “Fast-DC” Immunotherapy As a Potential Post-Remission Strategy for AML

Patients with acute myeloid leukemia (AML), not eligible for allogeneic hematopoietic stem cell transplantation (AHSCT), have a significant risk of disease relapse and death due to a lack of long-term disease control. To investigate the possibility of relapse prevention after initial chemotherapy, a safety and feasibility, single-center, open label, first in human phase I/II study using dendritic cells (DC) targeting PRAME and WT-1 was recently completed. Mature autologous DCs were rapidly (3-4 days) generated using full-length PRAME and WT-1 antigens and a maturation cocktail with a TLR 7/8 agonist. Intradermal vaccination of 2.5-5x106 WT-1 and 2.5-5x106 PRAME RNA-loaded DCs was performed at week 1, 2, 3, 4, 6 and then monthly for the remainder of a 2-year study period. Patients with WT1-positive AML, with or without PRAME positivity, had to be in morphological remission (+/- hematological recovery) after induction chemotherapy without being eligible for AHSCT. The study consisted of a phase I part (n=6) to assess early safety followed by a phase II part (n=14) which assessed further safety as well as clinical and immunological effects of vaccination. A total of 20 eligible patients (5 female and 15 male, ECOG score of 0) with a median age of 59 years (range 24 to 73) were included into the study. Risk groups based on HOVON/SAKK 102 criteria at screening identified 13 patients as good, 5 as intermediate and 2 as poor risk. All patients were positive for WT-1 and 17 for PRAME prior to chemotherapy. The mean time from first diagnosis to first vaccination was 10.1±3.7 months. Vaccinations were well tolerated, without any withdrawals from the study due to toxicity. No related serious or unexpected adverse events (AEs) were reported and the most common AEs were injection site related, accounting for 28% of all AEs, which were mild and transient in nature (Grade I; CTCAE v4.03). Grade I/II toxicities were experienced by 18/20 patients, whilst 5 (25%) experienced Grade III toxicity unlikely or not related to treatment. The 2-year survival probability from time of first vaccination was 80% (95% CI: 55-92). Importantly, patients at or above the age of 60 years (n=10) also had an 80% survival rate at 2 years (risk groups: 4 good, 4 intermediate, 2 poor). Nine patients relapsed under vaccination, of whom 4 died thereafter (three due to the underlying disease, one due to GvHD after transplantation) within the 2-year study duration, equating to a probability of progression free survival (PFS) of 55% (95% CI: 31-74). The PFS rate for the elderly patients (≥ 60 years) was 50%, while it was 60% for the younger patient group. Most relapses (5/9) appeared early, within 80 days after first vaccination. Out of the 9 relapsed patients, 6 could be successfully transplanted (104 to 380 days after first vaccination; risk groups: 4 good and 2 poor risk) and 4 were still alive at the end of the 2-year observation period. The DC manufacturing process reproducibly yielded a high number of DCs expressing the transfected tumor antigens, WT-1 or PRAME, as well as high surface levels of co-stimulatory molecules. After freeze-thawing, DCs were capable of CCL19-directed migration and, upon stimulation, secreted IL-12, but not IL-10. These phenotypic and functional characteristics indicate efficient DC maturation and activation despite the patients´ previous therapeutic regimens. DC properties were comparable irrespective of the patients' clinical outcome. Immune-monitoring of bulk T cells from vaccinated patients revealed that patients in stable remission had higher levels of HLA-DR-expressing T cells when compared to relapsing patients, both in peripheral blood and bone marrow. Such difference was observed prior to vaccination and throughout the entire treatment period, suggesting that T cell activation may play a role in maintaining remission. Analysis of 23 common AML mutations revealed that 6/9 relapsed patients had at least 1 mutation, with 4 patients having 3-6 mutations. In contrast, only 1 mutation already present at baseline was found in 4/11 patients in remission. Administration of autologous DC transfected with PRAME- and WT1-RNA is feasible, safe and well tolerated. The 2-year 80% survival rate, particularly in the elderly, and the 55% progression free survival warrant further studies to assess the efficacy of this vaccine approach in improving outcomes in patients with AML. Disclosures Raffegerst: Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company. Schnorfeil:Medigene Immunotherapies GmbH: Current Employment. Addo:Medigene Immunotherapies GmbH: Current Employment. Schendel:Medigene AG: Current Employment, Current equity holder in publicly-traded company. Pinkernell:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company.

Dynamic Changes in the Phenotype of Dendritic Cells in the Uterus and Uterine Draining Lymph Nodes After Coitus.

Dendritic cells (DCs) are essential for successful embryo implantation. However, the properties of uterine DCs (uDCs) during the implantation period are not well characterized. In this study, we investigated the dynamic changes in the uDC phenotypes during the period between coitus and implantation. In virgin mice, we evaluated the expressions of CD103 and XCR1, this is the first report to demonstrate uDCs expressing CD103 in XCR1+cDC1s and XCR1+cDC2s. On day 0.5 post coitus (pc), the number of uterine CD11c+CD103–MHC classIIhighCD86high–mature DCs rapidly increased and then decreased to non-pregnancy levels on days 1.5 and 2.5 pc. On day 3.5 pc just before implantation, the number of CD11c+CD103+MHC class IIdimCD86dim–immature DCs increased in the uterus. The increase in mature uDCs on day 1.5 pc was observed in both allogeneic- and syngeneic mating, suggesting that sexual intercourse, or semen, play a role in this process. Meanwhile, the increase in immature uDCs on day 3.5 pc was only observed in allogeneic mating, suggesting that allo-antigens in the semen contribute to this process. Next, to understand the turnover and migration of uDCs, we monitored DC movement in the uterus and uterine draining lymph nodes (dLNs) using photoconvertible protein Kikume Green Red (KikGR) mice. On day 0.5 pc, uDCs were composed of equal numbers of remaining DCs and migratory DCs. However, on day 3.5 pc, uDCs were primarily composed of migratory DCs, suggesting that most of the uDCs migrate from the periphery just before implantation. Finally, we studied the expression of PD-L2—which induces immunoregulation—on DCs. On day 3.5 pc, PD-L2 was expressed on CD103+-mature and CD103–-mature DCs in the uterus. However, PD-L2 expression on CD103–-immature DCs and CD103+-immature DCs was very low. Furthermore, both remaining and migratory DCs in the uterus and uterus-derived-DCs in the dLNs on day 3.5 pc highly expressed PD-L2 on their surface. Therefore, our study findings provide a better understanding of the dynamic changes occurring in uterine DCs and dLNs in preparation for implantation following allogeneic- and syngeneic mating.

Shaping of Dendritic Cell Function by the Metabolic Micro-Environment.

Nutrients are required for growth and survival of all cells, but are also crucially involved in cell fate determination of many cell types, including immune cells. There is a growing appreciation that the metabolic micro-environment also plays a major role in shaping the functional properties of dendritic cells (DCs). Under pathological conditions nutrient availability can range from a very restricted supply, such as seen in a tumor micro-environment, to an overabundance of nutrients found in for example obese adipose tissue. In this review we will discuss what is currently known about the metabolic requirements for DC differentiation and immunogenicity and compare that to how function and fate of DCs under pathological conditions can be affected by alterations in environmental levels of carbohydrates, lipids and amino acids as well as by other metabolic cues, including availability of oxygen, redox homeostasis and lactate levels. Many of these insights have been generated using in vitro model systems, which have revealed highly diverse effects of different metabolic cues on DC function. However, they also stress the importance of shifting toward more physiologically relevant experimental settings to be able to fully delineate the role of the metabolic surroundings in its full complexity in shaping the functional properties of DCs in health and disease.

Abstract 1021: A novel interaction between hydrogen peroxide metabolism and LDL-uptake controls immunergulatory properties in human dendritic cells - Potential implications for tumor immune escape

Abstract Introduction: Recent studies indicate that depletion of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) may actually allow tumors to evade immunological rejection prompting us to analyze the impact of H2O2-depletion on dendritic cells (DCs) as they play a pivotal role in tumor immunology. Methods: Primary human monocytes from healthy donors were differentiated/maturated to DCs w/o concomitant enzymatic H2O2-depletion (i.e. purified catalase). DCs were then analyzed for the expression of typical maturation-markers (i.e. CD80/86) and their functionality (i.e. induction/suppression of T-cell proliferation, secretion of distinct cytokines). To identify the underlying mechanisms for the differences observed we analyzed the role of H2O2- and cholesterol-metabolism as they are interconnected in monocyte-driven phagocytosis. Results: Depletion of H2O2 not only impaired DC-maturation (i.e. impaired upregulation of CD80/86), but additionally induced immunosuppressive properties such as suppression of T-cell proliferation via IDO, both typical features of so called regulatory DCs found in the tumor-microenvironment. However, the impact of H2O2-depletion throughout DC differentiation/maturation appears to be rather complex: Thus differentiated/matured DCs did not only feature properties of pro-tumorigenic macrophages (i.e. CD163- and PAI-1-expression) but also were capable of inducing IL10/IL17 double-expressing T-cells which have been reported to contribute to tumor-immune-escape in AML. Paradoxically, extracellular H2O2-depletion resulted in increased intracellular H2O2-levels, and increased LDL uptake, which has been reported to potentially affect immunological tumor rejection. Finally, we were able to establish a direct link between H2O2-production, LDL uptake, and induction of immunosuppressive DCs as both, inhibition of LDL uptake and depletion of intracellular H2O2, restored maturation and abrogated immunosuppressive properties. Conclusion: Alongside with earlier findings such as the ability of hepatic stromal cells to induce myeloid-derived suppressor cells via contact-dependent H2O2-depletion, the here presented study sheds new light on the role of H2O2 in tumor-immunology. Importantly, our findings unravel a so far unrecognized interaction between H2O2- and LDL-metabolism controlling distinct properties in DCs, which might in return explain the association between lipid metabolism and impaired immunity in some tumors. Bearing in mind that tumors employ several mechanisms to deplete ROS incl. H2O2 we believe that these findings may contribute to the development of novel (immune-) therapeutic approaches for cancer patients. Citation Format: Ann-Katrin Menzner, Tanja Rottmar, Simon Voelkl, Jacobus J. Bosch, Dimitrios Mougiakakos, Andreas Mackensen, Yazid J. Resheq. A novel interaction between hydrogen peroxide metabolism and LDL-uptake controls immunergulatory properties in human dendritic cells - Potential implications for tumor immune escape [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1021.