Maturation Stimuli Research Articles

TIM-3 expression on monocyte-derived dendritic cells

The T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), an inhibitory checkpoint receptor, has been identified as a crucial regulator of cellular immune responses. TIM-3 has been discovered as a receptor involved in the negative regulation of T cells. Recent studies have demonstrated that TIM-3 is expressed on innate immune cells, including dendritic cells (DCs), even at a higher level than T cells. In the tumor microenvironment, the majority of DCs have a monocytic origin. Models for studying such DCs in vitro are DC cultures generated from monocytes in the presence of growth factors. The present study aimed to investigate the expression of TIM-3 in IFNα-induced monocyte-derived DCs (IFN-DCs) and the impact of DC activation on TIM-3 expression. DCs were obtained by culturing the adherent fraction of mononuclear cells from healthy donors for 4 days in the presence of GM-CSF and IFNα, followed by LPS addition for 24 hours. Human double-stranded DNA (dsDNA, 5 μg/mL) was added as an activation stimulus to intact IFN-DCs at the stage of maturation, along with LPS. Expression of the membrane TIM-3 molecule was determined by flow cytometry, and the level of expression of TIM-3 mRNA – by real-time RT-PCR with reverse transcription. Intact donor IFN-DCs expressed the membrane TIM-3 molecule at a high level (more than 70% of cells). The addition of LPS as a maturation stimulus almost halved the expression of TIM-3 (pW < 0.05) without affecting the expression of HAVCR2/TIM-3 mRNA. Exogenous dsDNA (along with LPS) increased the expression of HAVCR2/TIM-3 mRNA by more than three times (pW = 0.05) with a decrease in the number of TIM-3+DCs (pW = 0.003). Our findings indicate the presence of mechanisms that support expression of this inhibitory checkpoint receptor under conditions of DC activation. Further studies of the regulation of TIM-3 expression by monocyte-derived dendritic cells will expand the understanding of the biological significance of inhibitory receptors on DCs from the point of view of the immune response, as well as, in the future, increase the effectiveness of current approaches in cancer immunotherapy using IFN-DCs and inhibitors of checkpoint molecules.

A versatile gemini amphiphile-based platform with STING-activating properties for efficient gene delivery into dendritic cells

Encouraging results from clinical trials have underscored the potential of gene-pulsed dendritic cell (DC) vaccines as a viable approach for immunotherapy. However, insufficient gene delivery and functional deficiencies in antigen presentation, migratory capacity and cytokine release of DC vaccines limited its broader clinical application. Here, a combinatorial design of cationic gemini amphiphile (GA) molecular library based on the four-component Ugi reaction (Ugi-4CR) has been developed to identify versatile non-viral vectors for gene-engineered DC vaccines. We demonstrated that the leading GA enabled robust transfection of plasmid DNA or mRNA into the DCs through the formation of minimalist binary complexes, which exhibited great advantages of low carrier/gene ratios, high gene loading efficiency and well biocompatibility. The identified GA stimulated strong type I interferon (IFN) expression via the intracellular stimulator of interferon genes (STING) pathway and enhanced DC maturation and activation, which could potentially facilitate strong immunogenicity of DC vaccines. Importantly, DC maturation and activation could be further strengthened by co-delivering maturation stimuli with mRNA/GA complexes, and adoptive transfer of gene-engineered DC vaccines elicited strong T cell responses and thus inhibited tumor growth in vivo. The further studies indicated GAs can be formulated into lipid nanoparticle (LNP)-like quinary complexes that presented 6.9-fold higher mRNA delivery efficiency on the DCs than the commercial Lipofectamine 2000. These results suggest that GA-based molecular library is a powerful platform to develop versatile gene delivery vectors for immune cell engineering.

Factor of time in dendritic cell (DC) maturation: short-term activation of DCs significantly improves type 1 cytokine production and T cell responses

Dendritic cells (DCs) have been intensively studied in correlation to tumor immunology and for the development DC-based cancer vaccines. Here, we present the significance of the temporal aspect of DC maturation for the most essential subsequent timepoint, namely at interaction with responding T cells or after CD40-Ligand restimulation. Mostly, DC maturation is still being achieved by activation processes which lasts 24 h to 48 h. We hypothesized this amount of time is excessive from a biological standpoint and could be the underlying cause for functional exhaustion. Indeed, shorter maturation periods resulted in extensive capacity of monocyte-derived DCs to produce inflammatory cytokines after re-stimulation with CD40-Ligand. This effect was most evident for the primary type 1 polarizing cytokine, IL-12p70. This capacity reached peak at 6 h and dropped sharply with longer exposure to initial maturation stimuli (up to 48 h). The 6 h maturation protocol reflected superiority in subsequent functionality tests. Namely, DCs displayed twice the allostimulatory capacity of 24 h- and 48 h-matured DCs. Similarly, type 1 T cell response measured by IFN-γ production was 3-fold higher when CD4+ T cells had been stimulated with shortly matured DC and over 8-fold greater in case of CD8+ T cells, compared to longer matured DCs. The extent of melanoma-specific CD8+ cytotoxic T cell induction was also greater in case of 6 h DC maturation. The major limitation of the study is that it lacks in vivo evidence, which we aim to examine in the future. Our findings show an unexpectedly significant impact of temporal exposure to activation signals for subsequent DC functionality, which we believe can be readily integrated into existing knowledge on in vitro/ex vivo DC manipulation for various uses. We also believe this has important implications for DC vaccine design for future clinical trials.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

A Bidens pilosa L. Non-Polar Extract Modulates the Polarization of Human Macrophages and Dendritic Cells into an Anti-Inflammatory Phenotype.

Different communities around the world traditionally use Bidens pilosa L. for medicinal purposes, mainly for its anti-inflammatory, antinociceptive, and antioxidant properties; it is used as an ingredient in teas or herbal medicines for the treatment of pain, inflammation, and immunological disorders. Several studies have been conducted that prove the immunomodulatory properties of this plant; however, it is not known whether the immunomodulatory properties of B. pilosa are mediated by its ability to modulate antigen-presenting cells (APCs) such as macrophages (MØs) and dendritic cells (DCs) (through polarization or the maturation state, respectively). Different polar and non-polar extracts and fractions were prepared from the aerial part of B. pilosa. Their cytotoxic and immunomodulatory effects were first tested on human peripheral blood mononuclear cells (PBMCs) and phytohemagglutinin (PHA)-stimulated PBMCs, respectively, via an MTT assay. Then, the non-cytotoxic plant extracts and fractions that showed the highest immunomodulatory activity were selected to evaluate their effects on human MØ polarization and DC maturation (cell surface phenotype and cytokine secretion) through multiparametric flow cytometry. Finally, the chemical compounds of the B. pilosa extract that showed the most significant immunomodulatory effects on human APCs were identified using gas chromatography coupled with mass spectrometry. The petroleum ether extract and the ethyl acetate and hydroalcoholic fractions obtained from B. pilosa showed low cytotoxicity and modulated the PHA-stimulated proliferation of PBMCs. Furthermore, the B. pilosa petroleum ether extract induced M2 polarization or a hybrid M1/M2 phenotype in MØs and a semi-mature status in DCs, regardless of exposure to a maturation stimulus. The immunomodulatory activity of the non-polar (petroleum ether) extract of B. pilosa on human PBMC proliferation, M2 polarization of MØs, and semi-mature status in DCs might be attributed to the low-medium polarity components in the extract, such as phytosterol terpenes and fatty acid esters.

An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

Abstract LB216: Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors

Abstract Suppressive myeloid cells inhibit antitumor immunity by preventing T cell responses. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. Our team recently identified fibronectin as a functional ligand for ILT3. The fibronectin-ILT3 interaction serves as a “stromal checkpoint” through which the extracellular matrix actively promotes myeloid cell suppression in the tumor microenvironment. We now report the pre-clinical characterization of NGM831, a humanized IgG1 (N297G) antibody that binds to ILT3 of human and cynomolgus monkey origin with high specificity and sub-nM affinity and blocks the interaction of ILT3 with both its reported ligands, fibronectin and ApoE. In a gene expression profiling experiment, treatment of tolerogenic dendritic cells with NGM831 in the presence of fibronectin decreased their expression of inhibitory and scavenger receptors (CD163, MRC1, MARCO, STAB1, LILRB2) and markers of dendritic cell immaturity (CD14, CD209, FOLR2) and increased their expression of genes involved in antigen presentation (TNFSF14, DCSTAMP, CD82, OLR1) and TH1 polarization (SPP1, TBX21), suggesting that these cells had been remodeled to a more stimulatory phenotype. Consistent with these observations, treatment of tolerogenic dendritic cells with NGM831 increased their production of pro-inflammatory cytokines in response to FcɣR ligation, restored their responsiveness to maturation stimuli such as lipopolysaccharide (LPS) and CD40 ligand (CD40L), and increased their production of chemokines that could induce the migration of other immune cells to sites of inflammation. In addition, NGM831 acted synergistically with pembrolizumab to increase the ability of dendritic cells to stimulate allogeneic T cells in mixed lymphocyte reactions. Taken together, these results demonstrate that NGM831 can block fibronectin-mediated immunosuppression and promote myeloid cell reprogramming. These data support the clinical development of the ILT3 antagonist antibody NGM831 alone and in combination with immune checkpoint blockade. NGM831 is expected to enter clinical trials for the treatment of advanced solid tumors in early 2022. Citation Format: Peirong Chen, Suzanne C. Crawley, Vicky Y. Lin, Varun Kapoor, Kevin J. Paavola, Hung-I Chen, Joshua S. Lichtman, Betty Li, Kalyani Mondal, Alan K. Kutach, Yan Wang, Daniel D. Kaplan, Julie M. Roda. Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB216.

Generation of high cross-presentation ability human dendritic cells by combination of interleukin 4, interferon β and GM-CSF.

Dendritic cell (DC)-based immunotherapies have been utilized for the treatment of numerous diseases. However, the conventional generation strategies of DCs in vitro require 7 days and these DCs showed an unsatisfactory function, which prompted us to explore new approaches. We found that in vitro culture of human CD14+ cells, in the medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, as well as interferon β (IFN-β) for 48 h, followed by the maturation stimuli of IL-1β and poly I:C for another 24 h can be differentiated into high cross-presentation ability DCs (G4B-DCs). These DCs express high levels of CD11c, CD86, and HLA-DR, producing a high level of tumor necrosis factor α (TNF-α). Of note, compared with the conventional DCs, G4B-DCs showed a higher ability to promote allogeneic naïve CD4+ T cell and CD8+ T cell proliferation and interferon (IFN)-γ production. These DCs also have the remarkable ability to induce Flu-M1-specific CD8+ T cells. In addition, we found that these G4B-DCs express partially the cDC1 phenotype. These data indicate that G4B-DC is unique and may provide a relatively rapid alternative method for potential clinical use.

Motor Performance in Male Youth Soccer Players: A Systematic Review of Longitudinal Studies.

The aim of this systematic review was to identify and synthesize the available information regarding longitudinal data addressing young soccer players’ motor performance changes. Following the Preferred Reporting Items for Systematic and Meta-analyses (PRISMA) statement, literature searches were performed in three databases: PubMed, ISI Web of Science and SCOPUS. The following descriptors were used: football, soccer, youth, young, player, athlete, physical performance, motor performance, longitudinal. The inclusion criteria were original articles in English with longitudinal data of young males (aged 10–18 years), with the aim to investigate motor performance serial changes. The initial search returned 211 records, and the final sample comprised 32 papers. These papers covered the European continent, and used mixed and pure longitudinal design with variation in sample size and age range. The reviewed studies tended to use different tests to assess the motor performance and aimed to identify changes in motor performance in several ways. In general, they indicated motor performance improvements with age, with a marked influence of biological maturity, body composition, and training stimuli. This review highlights the need for coaches and stakeholders to consider players’ motor performance over time whilst considering biological maturation, biological characteristics, and training stimuli.

In-Depth Analysis of the Impact of Different Serum-Free Media on the Production of Clinical Grade Dendritic Cells for Cancer Immunotherapy.

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

The Janus Kinase inhibitor tofacitinib impacts human dendritic cell differentiation and favours M1 macrophage development.

Several cytokines signalling via Janus Kinase (JAK) proteins have been implicated in the pathogenesis of immune-mediated inflammatory diseases, including psoriasis and rheumatoid arthritis (RA). Tofacitinib, a small JAK inhibitor, is approved for the treatment of RA and has demonstrated good efficacy in psoriasis phase III clinical trials. In this work, we analysed the in vitro effects of tofacitinib on the functions of human dendritic cells (DCs) and macrophages. When assessing the effects of tofacitinib on monocyte-derived DCs, we observed reduced differentiation of monocytes into immature DCs, as evidenced by a decreased transcription of CD209 and CD80. Phenotype assessment in the presence of tofacitinib suggested a switch towards a M1-like macrophage phenotype, as evidenced by the expression of M1 markers such as iNOS, as well as cytokines typically expressed by M1 cells, including IL-12 and IL-23. Of note, Arginase1 and CD200R, typically expressed by M2 cells, were absent on tofacitinib-treated DCs. Furthermore, tofacitinib affected the response of differentiated DCs to maturation stimuli such as LPS and IFNγ, resulting in a partial up-regulation of IL-23 and down-regulation of IL-12, as assessed by qPCR. When investigating macrophage development, we found that tofacitinib inhibited the ability of monocytes to differentiate and polarize into regulatory M2 macrophages, while rather enhancing the ability to develop into inflammatory M1-like macrophages, as evidenced by decreased expression of the M2 marker CD200R and enhanced production of IL-12 and IL-23. In conclusion, tofacitinib impacts the differentiation of human DCs and macrophages, it particularly favours generation of M1-like pro-inflammatory macrophages.

Mechanisms of Tolerance Induction through T Regulatory Cells during Chemotherapy-Mediated Immunogenic Cell Death in Acute Myeloid Leukemia

Background: Although the recent advances have allowed the discovery of new and less toxic forms of treatment for acute myeloid leukemia (AML), chemotherapy remains as one of the most used treatments. Some chemotherapeutic agents can induce a type of cell death - immunogenic cell death (ICD) - that can promote modifications in cancer cells, which activates immune system against leukemia cells. Even though this can help the immune system fighting the cancer, chemotherapy is also responsible for inducing tolerogenic mechanisms, that enable cancer cells to escape from immune system control. We previously demonstrated that enzyme indoleamine-2,3-dioxygenase-1 (IDO1) is involved in T regulatory cells (Tregs) induction in AML during ICD induced by chemotherapy. Dying leukemia cells release ATP which binds P2X7 receptor on dendritic cells (DCs), resulting in IDO1 up-regulation. In the same time, ATP can be catabolized by CD39 e CD73 ectonucleotidases expressed on DCs in adenosine with suppressive properties, thus resulting in the stabilization of Tregs phenotype and function. In the present work, we have investigated the role of ATP catabolism in Tregs induction after chemotherapy treatment in AML. Materials and methods: For in vitro experiments,CD14 and CD3 positive cells were obtained from peripheral blood mononuclear cells of healthy donors. HL-60 leukemia cell line cells, untreated or treated with daunorubicin (DNR; 500 ng/ml) or cytarabine (Ara-C; 20 µg/ml) were loaded in immature DCs, previously differentiated from monocytes. After 24 h, immature DCs and DCs matured by treated HL-60 cells or DCs treated with ATP in presence or absence of P2X7 inhibitor, were analyzed for phenotype and used to activate T cells. After 5 days, a detailed phenotype characterization of induced Tregs was performed by flow cytometry. For in vivo experiments, Balbc/J wt mice were subcutaneously injected with 2x106 WEHI-3B leukemia cells. DNR (3 mg/kg), Ara-C (50 mg/kg) or sterile PBS vehicle (placebo) were administered at post inoculum days 7 and 9, then the tumor infiltrating lymphocytes were purified from tumor mass and stained by flow cytometry. Results: Firstly, we in vitro characterized immune phenotype of DCs loaded with HL-60 cells, previously treated with DNR, Ara-C or medium alone. We identified DNR as a potent maturation stimulus as compared to DCs loaded with untreated cells or unloaded immature DCs. In particular, both DNR and Ara-C induced a significant up-regulation of CD83, CD80 and CD86, but only DNR induced a significant up-regulation of CCR7, which is required for DC migration to lymphonodes. Then, the effect of chemotherapy on CD39 and CD73 expression in DCs was also evaluated. DNR induced a significant up-regulation of CD73-the rate limiting step of ATP catabolism, whereas upon Ara-C treatment there was a trend in the up-regulation of both CD39 and CD73. Interestingly, DCs treated with ATP alone achieved similar maturation markers level as DNR, but showed neither CCR7 nor CD39/CD73 up-regulation, which suggests that ATP alone is not sufficient to activate the ATP catabolism pathway. The analysis of Tregs induced after 5 days of co-culture with DCs treated as previously described showed a significant up-regulation of "fitness markers", in particular OX40/CD39 after DNR and OX40/PD-1 after DNR and Ara-C treatment as compared to unloaded immature DCs. Mouse models confirmed the capacity of DNR and Ara-C to induce DCs maturation and activation of ATP catabolism pathways by significantly inducing CD39 and CD73 up-regulation. Moreover, chemotherapy treatment resulted in the stabilization of Tregs suppressive phenotype as demonstrated by the up-regulation of OX40 and ICOS fitness markers in comparison to placebo treatment. Conclusions: Our data suggest that different mechanisms operate in tumor microenvironment. IDO may induce Tregs in an ATP-dependent manner, whereas the ATP catabolism may be involved in the stabilization of Tregs suppressive phenotype. In this context, the specific role of the main bioproduct of ATP catabolism, adenosine, is under investigation. Our results can help to understand the mechanisms of tolerance induction after chemotherapy in AML. Disclosures Cavo: takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.

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.

Matured Tolerogenic Dendritic Cells Effectively Inhibit Autoantigen Specific CD4+ T Cells in a Murine Arthritis Model.

Tolerogenic dendritic cells (tolDCs) are a promising treatment modality for diseases caused by a breach in immune tolerance, such as rheumatoid arthritis. Current medication for these diseases is directed toward symptom suppression but no real cure is available yet. TolDC-based therapy aims to restore immune tolerance in an antigen-specific manner. Here we used a mouse model to address two major questions: (i) is a maturation stimulus needed for tolDC function in vitro and in vivo and is maturation required for functioning in experimental arthritis and (ii) can tolDCs modulate CD4+ T cell responses? To answer these questions, we compared matured and immature dexamethasone/vitamin D3-generated tolDCs in vitro. Subsequently, we co-transferred these tolDCs with naïve or effector CD4+ T cells to study the characteristics of transferred T cells after 3 days with flow cytometry and Luminex multiplex assays. In addition, we tested the suppressive capabilities of tolDCs in an experimental arthritis model. We found that tolDCs cannot only modulate naïve CD4+ T cell responses as shown by fewer proliferated and activated CD4+ T cells in vivo, but also effector CD4+ T cells. In addition, Treg (CD4+CD25+FoxP3+) expansions were seen in the proliferating cell population in the presence of tolDCs. Furthermore, we show that administered tolDCs are capable to inhibit arthritis in the proteoglycan-induced arthritis model. However, a maturation stimulus is needed for tolDCs to manifest this tolerizing function in an inflammatory environment. Our data will be instrumental for optimization of future tolDC therapies for autoimmune diseases.

Exploratory analysis of beta thalassemia and aplastic anemia transcriptomes for the advancement of ex situ erythroblast proliferation and maturation.

Background & Aim Blood supply shortage is emerging as an issue worldwide, partially due to an aging and shrinking population which diminishes the healthy blood donor pool available to national blood programmes. To secure a safe and consistent supply of blood, various strategies have been explored. With development of various molecular tools, ex vivo generation of RBCs is emerging as a promising technology to alleviate the blood supply shortage issue. Although much progress has been made in the production of enucleated RBCs from stem cells, further research is required to optimize the scalability of enucleated RBC production in a culture system, with emphasis on advanced studies into proliferation and maturation stimuli for erythropoetic progenitors. Meanwhile, the contrasting stories of erythroid hyperplasic anemia and aplastic anemia presents an interesting topic to investigate the underlying mechanisms behind erythroid cell proliferation and maturation. Beta thalassemia is caused by a defective Beta globin gene that destabilizes alpha-tetrameric formation in erythroid cells and causes premature cell death. The continued stimulation of ineffective erythropoiesis leads to erythroid hyperplasic anemia. Conversely, aplastic anemia is a condition characterized by massive erythroid cell depletion in the marrow. While both conditions present anemia clinically, the cascade of events leading up to anemia is different for each case, resulting in two dissimilar marrow microenvironments. Thus, we hypothesized that through studying the differential expressional patterns of progenitors from each condition, we can determine the important mechanistic pathways underlying erythropoiesis and erythroid cell maturation. Methods, Results & Conclusion In our study, we sought to discover the differential gene expression between Beta Thalassemia and Aplastic Anemia. To that end, we obtained the transcriptomic data of erythroid progenitor cells of Beta Thalassemia and Aplastic anemia, from publicly available databases. We performed exploratory gene expressional analysis to identify differentially expressed genes in each condition. Pathway analysis was then conducted on each set of differentially expressed genes to identify significantly perturbed signalling nodes. These genes and pathways were compared against known literature on erythroid cell proliferation and maturation, to identify novel genes and pathways in our analysis. Future work will test the application of these genes in ex vivo RBC production.

Substrate Stiffness, Cell Anisotropy, and Cell-Cell Contact Contribute to Enhanced Structural and Calcium Handling Properties of Human Embryonic Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can be utilized to understand the mechanisms underlying the development and progression of heart disease, as well as to develop better interventions and treatments for this disease. However, these cells are structurally and functionally immature, which undermines some of their adequacy in modeling adult heart tissue. Previous studies with immature cardiomyocytes have shown that altering substrate stiffness, cell anisotropy, and/or cell-cell contact can enhance the contractile and structural maturation of hPSC-CMs. In this study, the structural and calcium handling properties of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were enhanced by exposure to a downselected combination of these three maturation stimuli. First, hESC-CMs were seeded onto substrates composed of two commercial formulations of polydimethylsiloxane (PDMS), Sylgard 184 and Sylgard 527, whose stiffness ranged from 5 kPa to 101 kPa. Upon analyzing the morphological and calcium transient properties of these cells, it was concluded that a 21 kPa substrate yielded cells with the highest degree of maturation. Next, these PDMS substrates were microcontact-printed with laminin to force the cultured cells into rod-shaped geometries using line patterns that were 12, 18, or 24 μm in width. We found that cells on the 18 and 24 μm pattern widths had structural and functional properties that were superior to those on the 12 μm pattern. The hESC-CMs were then seeded onto these line-stamped surfaces at a density of 500 000 cells per 25-mm-diameter substrate, to enable the formation of cell-cell contacts at their distal ends. We discovered that this combination of culture conditions resulted in cells that were more structurally and functionally mature than those that were only exposed to one or two stimuli. Our results suggest that downselecting a combination of mechanobiological stimuli could prove to be an effective means of maturing hPSC-CMs in vitro.

Extracellular vesicles from mature dendritic cells (DC) differentiate monocytes into immature DC.

During inflammation, murine and human monocytes can develop into dendritic cells (DC), but this process is not entirely understood. Here, we demonstrate that extracellular vesicles (EV) secreted by mature human DC (maDC) differentiate peripheral monocytes into immature DC, expressing a unique marker pattern, including 6-sulfo LacNAc (slan), Zbtb46, CD64, and CD14. While EV from both maDC and immature DC differentiated monocytes similar to GM-CSF/IL-4 stimulation, only maDC-EV produced precursors, which upon maturation stimulus developed into T-cell-activating and IL-12p70-secreting maDC. Mechanistically, maDC-EV induced cell signaling through GM-CSF, which was abundant in EV as were IL-4 and other cytokines and chemokines. When injected into the mouse skin, murine maDC-EV attracted immune cells including monocytes that developed activation markers typical for inflammatory cells. Skin-injected EV also reached lymph nodes, causing a similar immune cell infiltration. We conclude that DC-derived EV likely serve to perpetuate an immune reaction and may contribute to chronic inflammation.

GM-CSF Culture Revisited: Preparation of Bulk Populations of Highly Pure Dendritic Cells from Mouse Bone Marrow.

Dendritic cells (DC) loaded with specific peptides are strongly immunogenic for T cells and can be used for cancer immunotherapy. For immunogenic tumors such as melanoma, injection of autologous DC loaded with tumor cell extracts or peptides can induce tumor regression but in only a small proportion of patients. Nevertheless, recent studies on the efficacy of checkpoint blockade for boosting antitumor immunity plus advances in defining tumor neoantigens are stimulating renewed interest in DC immunotherapy. Despite intensive investigation, however, preparation of bulk populations of mature DC has proved difficult, and most preparations contain a significant proportion of potentially tolerogenic immature DC. In this study, we have modified the well-established GM-CSF culture system to prepare substantial quantities of highly pure (>95%) mature DC from mouse bone marrow cells and defined their progenitors. We show that obtaining high yields and purity of DC are heavily dependent on cell density in the cultures and the tempo of addition of growth and maturation stimuli. When loaded with specific peptide, the DC are strongly immunogenic for CD4 and CD8 T cells in vivo and elicit effective antitumor immunity.

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.