Maturation Of Monocyte-derived Dendritic Cells Research Articles

GMP-compliant human monocyte-derived dendritic cells for cancer vaccination generated by using the Quantum® hollow fiber bioreactor system

Abstract Background The Quantum® hollow fiber bioreactor system represents a platform integrating GMP-compliant manufacturing steps in a closed system for automated cultivation of cellular products. A previous report to generate monocyte-derived DCs (Mo-DCs) for cancer immunotherapy involved fibronectin-coating of the hollow-fibers and trypsin digestion to harvest cells and proved unsatisfactory as only one tenth of an apheresis product could be processed. Thus, optimization of this approach is needed. Methods Monocytes were enriched by using the Elutra® cell separation system and were differentiated over 6 days into immature DC in the presence of GM-CSF + IL-4, and then exposed to a maturation cocktail to generate mature Mo-DCs. This was performed in parallel either by using the Quantum® or by our in-house established standard protocol in cell culture bags. Phenotype, antigen presentation, and functionality of these Mo-DCs were then analyzed and compared. Results Intitial tests with the Quantum® were performed to avoid the initial fribronectin coating and trypsin digestion for harvesting of cells, which required optimization of media exchange rate, cytokine concentration and cytokine addition. Under optimized conditions, cells cultured in the Quantum® resulted in a yield of 27.8% mature DCs (related to input monocytes) with CD83 expression in 92.0% of these cells. Total yield of mature Mo-DCs was slightly higher when the in-house established standard protocol was used. Survival of Mo-DCs analyzed in the washout test (24 hour culture in medium without cytokines) was comparable with both methods. Cell surface CD80, CD83, CD86, and HLA-DR expression was in general higher in DCs generated by our standard protocol. GFP-electroporated Mo-DCs generated by Quantum®, however, showed a trend towards higher GFP- expression as well as towards higher T-cell stimulation and proliferation in the primary allogeneic MLR-assay. Conclusion We have adapted the Quantum® system to process one complete apheresis product to yield a large number of mature and functional GMP-compliant monocyte-derived DCs for the use in cancer immunotherapy without any need for fibronectin coating or trypsin digestions. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Photodynamic Modulation of Type 1 Interferon Pathway on Melanoma Cells Promotes Dendritic Cell Activation.

The immune response against cancer generated by type-I-interferons (IFN-1) has recently been described. Exogenous and endogenous IFN-α/β have an important role in immune surveillance and control of tumor development. In addition, IFN-1s have recently emerged as novel DAMPs for the consecutive events connecting innate and adaptive immunity, and they also have been postulated as an essential requirement for induction of immunogenic cell death (ICD). In this context, photodynamic therapy (PDT) has been previously linked to the ICD. PDT consists in the administration of a photosensitizer (PS) and its activation by irradiation of the affected area with visible light producing excitation of the PS. This leads to the local generation of harmful reactive oxygen species (ROS) with limited or no systemic defects. In the current work, Me-ALA inducing PpIX (endogenous PS) was administrated to B16-OVA melanoma cells. PpIX preferentially localized in the endoplasmic reticulum (ER). Subsequent PpIX activation with visible light significantly induced oxidative ER-stress mediated-apoptotic cell death. Under these conditions, the present study was the first to report the in vitro upregulation of IFN-1 expression in response to photodynamic treatment in melanoma. This IFN-α/β transcripts upregulation was concurrent with IRF-3 phosphorylation at levels that efficiently activated STAT1 and increased ligand receptor (cGAS) and ISG (CXCL10, MX1, ISG15) expression. The IFN-1 pathway has been identified as a critical molecular pathway for the antitumor host immune response, more specifically for the dendritic cells (DCs) functions. In this sense, PDT-treated melanoma cells induced IFN-1-dependent phenotypic maturation of monocyte-derived dendritic cells (DCs) by enhancing co-stimulatory signals (CD80, MHC-II) and tumor-directed chemotaxis. Collectively, our findings showed a new effect of PDT-treated cancer cells by modulating the IFN-1 pathway and its impact on the activation of DCs, emphasizing the potential relevance of PDT in adoptive immunotherapy protocols.

Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

Automated Good Manufacturing Practice–compliant generation of human monocyte-derived dendritic cells from a complete apheresis product using a hollow-fiber bioreactor system overcomes a major hurdle in the manufacture of dendritic cells for cancer vaccines

BackgroundAlthough dendritic cell (DC)–based cancer vaccines represent a promising treatment strategy, its exploration in the clinic is hampered due to the need for Good Manufacturing Practice (GMP) facilities and associated trained staff for the generation of large numbers of DCs. The Quantum bioreactor system offered by Terumo BCT represents a hollow-fiber platform integrating GMP-compliant manufacturing steps in a closed system for automated cultivation of cellular products. In the respective established protocols, the hollow fibers are coated with fibronectin and trypsin is used to harvest the final cell product, which in the case of DCs allows processing of only one tenth of an apheresis product. Materials and ResultsWe successfully developed a new protocol that circumvents the need for fibronectin coating and trypsin digestion, and makes the Quantum bioreactor system now suitable for generating large numbers of mature human monocyte-derived DCs (Mo-DCs) by processing a complete apheresis product at once. To achieve that, it needed a step-by-step optimization of DC-differentiation, e.g., the varying of media exchange rates and cytokine concentration until the total yield (% of input CD14+ monocytes), as well as the phenotype and functionality of mature Mo-DCs, became equivalent to those generated by our established standard production of Mo-DCs in cell culture bags. ConclusionsBy using this new protocol for the Food and Drug Administration–approved Quantum system, it is now possible for the first time to process one complete apheresis to automatically generate large numbers of human Mo-DCs, making it much more feasible to exploit the potential of individualized DC-based immunotherapy.

Cinnamon extract inhibits allergen-specific immune responses in human and murine allergy models.

Ceylon cinnamon has been shown to possess anti-inflammatory properties in many diseases including allergic inflammation. The aim of this study was to analyse in more detail the effects of cinnamon extract (CE) and its major compounds p-cymene and trans-cinnamaldehyde (CA) on allergen-specific immune responses in vitro and in vivo. Therefore, monocyte-derived mature dendritic cells (DC) from grass or birch pollen allergic donors were pulsed with the respective allergen in the presence or absence of CE, p-cymene, CA or the solvent ethanol and co-cultured with autologous CD4+ T cells. Furthermore, basophil activation test was performed with or without CE or ethanol treatment. For the in vivo experiments, BALB/c mice were immunized with ovalbumin (OVA) and orally treated with CE or ethanol. Addition of CE, p-cymene or CA, but not ethanol significantly inhibited DC maturation and subsequent allergen-specific T cell proliferation as well as Th1 and Th2 cytokine production. Sulphidoleukotriene release and CD63 expression by basophils were also significantly diminished after addition of CE. In vivo, treatment of OVA-sensitized mice with CE led to a significant shift from OVA-specific IgE towards IgG2a production and to a strong inhibition of OVA-specific proliferation. Moreover, airway inflammation as well as anaphylaxis after intranasal or systemic allergen challenge was significantly reduced in CE-treated mice. Furthermore, topical application of CE prevented calcipotriol-induced atopic dermatitis-like inflammation in these mice. Taken together, our data indicate that the anti-inflammatory effect of cinnamon might be exploited for treatment of allergic inflammation, which needs to be further investigated.

A Functional Assay to Determine the Capacity of Oncolytic Viruses to Induce Immunogenic Tumor Cell Death.

Oncolytic immunotherapy efficacy relies partially on the induction of immunogenic tumor cell death following infection with oncolytic viruses (OV) to induce an antitumor immune response. Here, we describe a method to determine if an OV is able to induce such an immunogenic tumor cell death. This method consists in testing whether tumor cells lysed by an OV are able to induce the maturation of human monocyte-derived immature dendritic cells (Mo-iDC).

Abstract 2340: Differentiation of pro- and anti-inflammatory features of monocyte-derived dendritic cells

Abstract Background: Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are consequently exploited in cancer immunotherapy in order to present tumor-associated antigens to lymphocytes. The ability of tumor cells to induce tolerogenic DCs and avoid the immune system challenges DC-based immunotherapies. Utilizing buffycoats of healthy blood donors we have investigated the activation of gene expression programs conventionally associated with either pro- or anti-inflammatory features of monocyte-derived DCs. Methodology: Following isolation of monocytes from buffycoats, various culture conditions were tested to generate immature DCs and to mature and activate them in vitro. Genome-wide gene expression, secretion of cytokines and tryptophan metabolites, surface expression of maturation and activation markers, and functional abilities were analyzed. Specifically, beta-catenin and indoleamine 2,3-dioxigenase 1 (IDO-1) activation were examined using both agonists and inhibitors of those pathways. Results: A considerable variability among buffycoats of different healthy blood donors was observed regarding DC yield and cytokine secretion. Induction time to generate immature DCs with interleukin-4 and granulocyte-macrophage colony-stimulating factor affected DC differentiation. Several maturation conditions induced strong IDO-1 expression. Activation of beta-catenin during DC maturation induced anti-inflammatory features that could be competed by adding beta-catenin inhibitors during cultivation of the cells. Pro- and anti-inflammatory features often co-existed in mature DCs. Conclusions: Beta-catenin and IDO-1 activation are prevalent according to different conditions of monocyte-derived DC maturation and associated with anti-inflammatory features. Both pathways can be inhibited by specific antagonists. More robust pro-inflammatory DC generation is desirable for more effective cancer immunotherapy. Citation Format: Ragnhild Maukon Bakke, Waqas Azeem, Silke Appel, Karl-Henning Kalland, Anne Margrete Oyan. Differentiation of pro- and anti-inflammatory features of monocyte-derived dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2340.

Thymic stromal lymphopoietin epigenetically upregulates Fc receptor γ subunit–related receptors on antigen-presenting cells and induces TH2/TH17 polarization through dectin-2

Fc receptor γ subunit (FcRγ)-related receptors expressed on antigen-presenting cells (APCs) enhance allergen sensitization and allergic inflammation. DNA demethylation of the high-affinity IgE receptor γ subunit gene (FCER1G) leads to FcRγ and FcεRI overexpression on monocytes from patients with atopic dermatitis. We investigated epigenetic mechanisms underlying FCER1G demethylation and upregulation of FcRγ-related receptors on APCs and the consequent effect on allergic responses. Effects of thymic stromal lymphopoietin (TSLP) on expression of FcRγ and its related receptors and methylation or hydroxymethylation of FCER1G in human monocytes were assessed. Recruitment of ten-eleven translocation protein (TET) 2 to FCER1G by TSLP-activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) was evaluated. Effects of TSLP on expression of FcRγ-related receptors and costimulatory receptors on monocyte-derived dendritic cells (DCs) and the ability of DCs to take up ovalbumin were analyzed. TSLP-induced TH polarization and related cytokine production were also analyzed. pSTAT5 activation by TSLP resulted in TET2 recruitment to FCER1G, leading to FCER1G demethylation and subsequent upregulation of FcRγ-related receptors on monocytes. TSLP not only stimulated monocyte-derived DC maturation but also maintained their allergen uptake ability, likely through maintenance and upregulation of FcRγ-related receptors. Allergen sensitization and upregulation of TH2/TH17-related cytokines contributed to TSLP-DC-induced TH2/TH17 polarization. The latter was attenuated on neutralization with a dectin-2 antibody. TSLP mediated upregulation of FcRγ-related receptors on APCs through activation of pSTAT5, which recruited TET2 to induce FCER1G demethylation. TSLP-induced allergic TH2/TH17 polarization likely depends on dectin-2-mediated allergen sensitization and upregulation of TH2/TH17-related cytokines.

PF446 EVALUATING EX VIVO EXPANSION OF FUNCTIONALLY COMPETENT MULTI-SPECIFIC T LYMPHOCYTES TO MINOR HISTOCOMPATIBILITY ANTIGEN FOR THE TREATMENT OF HEMATOLOGICAL MALIGNANCIES

Background: Allogeneic hematopoietic stem cell transplantation represents the sole curative treatment for high risk and relapsing leukemia. The presence of donor T cells in the graft mediates the powerful graft-versus-leukemia (GvL) effect. Recognition of minor histocompatibility antigen (MiHA) by T cells forms the basis of this GvL effect. MiHAs are small major histocompatibility antigen-restricted peptides encoded by germline polymorphism. Their preferential tissue restriction makes them an excellent target for adoptive T cell therapy for hematological malignancies. However, this form of anti-cancer activity is highly toxic due to the induction of detrimental graft-versus-host-disease mediated by donor T cells that recognize MiHAs expressed on non-hematopoietic cells. Thus, there is a scope for improving this form of adoptive T cell therapy by pre-activating and expanding donor-derived-T lymphocytes against specific MiHAs that are predominantly expressed on hematopoietic cells and may lead to improved cancer immunotherapy for hematologic cancers. Aims: The aim of this study was to evaluate the ex vivo expansion protocol of functionally competent MiHA specific CD8+ T cells. As an alternative to clinical studies where monocyte-derived dendritic cells (Mo-DC) are pulsed with single MiHA peptide, we propose an approach where Mo-DC pulsed with several MiHA peptides are used to activate and expand MiHA specific CD8+ T cells. We hypothesized that priming CD8+ T cells with multiple MiHAs may result in a multi-specificity product consisting of an oligoclonal T cell population reactive to multiple MiHAs and/or a dominant T cell response to one of the MiHAs. Further, we aim to understand potentially differential MiHA immunogenicity by quantifying MiHA presentation by dendritic cells using labeled-MiHAs. Methods: Mature Mo-DC were pulsed with single or multiple MiHA peptides, irradiated and co-cultured with autologous peripheral blood mononuclear cells for 21 days. Expansion of MiHA specific T cells was evaluated using dextramer staining. The functionality of antigen-specific T cells was assessed by interferon-γ (IFN-γ) and tumor necrosis factor-α production, CD107a degranulation, and IFN-γ ELISpot. In parallel, labeled-MiHA presentation by Mo-DC was quantified using flow cytometry. Results: In this study, we concluded on the feasibility of using Mo-DC pulsed with multiple peptides to expand MiHA specific T cells. Importantly, in several experiments, we also observed CD8+ T cells reactive to more than one MiHA demonstrating multiple antigen-specific responses within the same culture. However, variability was observed in the magnitude of this response. This variability might rely, in part, on the MiHA presentation by Mo-DC; Mo-DCs pulsed with four or more peptides resulted in higher cell mortality and had lower expression of the costimulatory molecule CD80, thus suggesting that pulsing Mo-DCs with four or more MiHAs may impact the antigen-presenting capacity of these cells. Interestingly, we demonstrated, using labeled-MiHAs that the peptides were presented by Mo-DC at variable levels. Summary/Conclusion: Our results demonstrate that we could generate a multi-specific T cell response using Mo-DC pulsed with multiple MiHA peptides. Ongoing experiments will shed light on the variability seen in the antigen presentation by Mo-DC derived from different donors and peptides used. Understanding the fundamental biology of MiHA specific T cells for cancer immunotherapy will allow us to develop a personalized approach for the treatment of high-risk leukemia patients.

Expression of the Tim3-galectin-9 axis is altered in drug-induced maculopapular exanthema.

Drug-induced maculopapular exanthemas (MPEs) are mediated by Th1 CD4+ T cells. One of the mechanisms of control of Th1 cells in homeostasis is the interaction between the checkpoint inhibitor Tim3 and its physiological ligand galectin-9 (Gal9). Disorders affecting this axis may be responsible for various autoimmune and immunological diseases. The aim of this study was to determinate the influence of the Tim3-Gal9 axis on the development of MPE induced by drugs. Frequencies of different cell subsets and the expression of Tim3 and Gal9 were measured in peripheral blood by flow cytometry and in skin biopsies by immunohistochemistry. Gal9 expression was assessed by RT-qPCR; its release was measured by multiplex assay. The effects of blocking or enhancing the Tim3-Gal9 axis on monocyte-derived dendritic cell (moDC) maturation and T-cell proliferation were determined by flow cytometry. The expression of Tim3 was significantly reduced in peripheral blood Th1 cells and in the skin of MPE patients vs controls. Gal9 expression and release were significantly reduced in patient peripheral blood and moDCs, respectively. The addition of exogenous Gal9 significantly reduced Tim3+ Th1 proliferation, although Treg proliferation increased. This study showed the involvement of the Tim3-Gal9 axis in MPE. The reduced expression of Tim3 in Th1 cells together with the impaired expression of Gal9 in PBMCs and DCs appears to have a role in the development of the disease. The potential of Gal9 to suppress Th1 and enhance Treg proliferation makes it a promising tool for treating these reactions.

Δ42PD-1 enhances HIV replication and suppresses maturation of dendritic cells

Abstract Previously, we discovered a new PD-1 isoform, which consists of a 42-base pairs loss from the start of exon 2 that is equivalent to a 14 amino acid in-frame deletion. Therefore, we named this new PD-1 isoform as Δ42PD-1. Here, we found that Δ42PD-1 was highly expressed on monocyte-derived dendritic cell (MoDC) in healthy donors, while significantly decreased in HIV patients with or without antiretroviral therapy. Consistently, in vitro assay, HIV infection suppressed Δ42PD-1 expression in total MoDCs. However, upregulation of Δ42PD-1 was observed in mature MoDC (high CD83+CD86+) after HIV infection, although the mature MoDC population was unraised, which consistent with previous report that HIV infection do not mature MoDC. Therefore, to investigate the role of Δ42PD-1 on HIV replication, Δ42PD-1 positive and negative MoDC were sorted out by FACS and infected with R5-tropic HIV (JR-CSF) respectively. Interestingly, Δ42PD-1 positive MoDC was higher susceptible to HIV than Δ42PD-1 negative MoDC. Maturation is important for dendritic cell to perform innate and adaptive immune response. Further investigation revealed that Δ42PD-1 negative MoDC had higher maturation level than Δ42PD-1 positive MoDC after LPS stimulation, which means that Δ42PD-1 may suppress DC maturation. So far, we conclude that Δ42PD-1 not only enhances HIV replication, but also suppresses dendritic cell maturation. It warrants to further study the role of Δ42PD-1 on DC-mediated immune responses during HIV infection in vivo.

Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro

BackgroundHepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood.MethodsHere, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry.ResultsThe IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose–dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered.ConclusionsOur findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.

The effect of age on foal monocyte-derived dendritic cell (MoDC) maturation and function after exposure to killed bacteria

Neonatal foals are uniquely susceptible to certain infections early in life. Dendritic cells (DC) are vital in the transition between the innate and adaptive immune response to infection, but DC biology in foals is not fully characterized. Monocyte-derived DC represent a suitable in vitro model similar to DC that differentiate from monocytes recruited from circulation. We hypothesized that foal monocyte-derived DC (MoDC) would exhibit age-dependent phenotypic and functional differences compared to adult horse MoDC. MoDC generated from 9 horses (collected once) and from 8 foals (collected at 1, 7, and 30 days-of-age) were exposed to killed whole cell Escherichia coli or Staphylococcus aureus bacteria. MoDC expression of MHC class II (MHC class-II), CD86, and CD14 were measured by flow cytometry, and supernatant cytokine concentrations of IL-4, IL-17, IFN-γ, and IL-10 were quantified with a validated immunoassay. The percentage of MoDC expressing MHC class-II and CD86 was lower and CD14 was higher for cells generated from 1-day-old foals compared to cells generated from adult horses (P < 0.0001). Bacterial exposure increased the percentage of cells expressing CD86 at all ages (P < 0.0001). Bacteria-exposed MoDC from 1-day-old foals produced significantly less IL-4, IL-17, and IFN-γ than adult MoDC produced in response to bacterial exposure (P ≤ 0.04). Following bacterial exposure, foal MoDC phenotype and cytokine secretion were different than those of mature horses. These differences could reduce the ability of foals to generate a protective immune response against bacterial infection.

D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells.

Mutations in isocitrate dehydrogenase (IDH) or a reduced expression of L-2-hydroxyglutarate (HG)-dehydrogenase result in accumulation of D-2-HG or L-2-HG, respectively, in tumor tissues. D-2-HG and L-2-HG have been shown to affect T-cell differentiation and activation; however, effects on human myeloid cells have not been investigated so far. In this study we analyzed the impact of D-2-HG and L-2-HG on activation and maturation of human monocyte-derived dendritic cells (DCs). 2-HG was taken up by DCs and had no impact on cell viability but diminished CD83 expression after Lipopolysaccharides (LPS) stimulation. Furthermore, D-2-HG and L-2-HG significantly reduced IL-12 secretion but had no impact on other cytokines such as IL-6, IL-10 or TNF. Gene expression analyses of the IL-12 subunits p35/IL-12A and p40/IL-12B in DCs revealed decreased expression of both subunits. Signaling pathways involved in LPS-induced cytokine expression (NFkB, Akt, p38) were not altered by D-2-HG. However, 2-HG reprogrammed LPS-induced metabolic changes in DCs and increased oxygen consumption. Addition of the ATP synthase inhibitor oligomycin to DC cultures increased IL-12 secretion and was able to partially revert the effect of 2-HG. Our data show that both enantiomers of 2-HG can limit activation of DCs in the tumor environment.

Human Leukocyte Antigen-G Restores Immune Tolerance in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder in which loss of immune tolerance leads to platelet destruction. Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class I antigen including four membrane-bound (mHLA-G, HLA-G1 to -G4) and three soluble (sHLA-G, HLA-G5 to -G7) isoforms due to alternative splicing of the HLA-G primary transcript. HLA-G exerts immunosuppressive functions by interacting with its inhibitory receptors, immunoglobulin-like transcripts (ILTs), expressing differentially on natural killer cells, T cells, and antigen presenting cells.Upon binding to ILTs, HLA-G suppressed lymphocyte proliferation, promoted apoptosis of cytotoxic lymphocytes and natural killer cells, inhibited antibody secretion from activated B cells, and induced expansion of suppressive T cells, tolerogenic dendritic cells and myeloid-derived suppressor cells. HLA-G supports immune escape, and high level of HLA-G is associated with unfavorable outcomes in cancer patients. Meanwhile, HLA-G-induced tolerance can benefit human allotransplantation and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, but the role of HLA-G in the treatment of ITP is still unclear.Here we observed significantly less soluble HLA-G in plasma from ITP patients positive for anti-glycoprotein (GP) IIb/IIIa and/or anti-GPIb/IX autoantibodies compared with autoantibody-negative patients or healthy controls. However, no significant difference was found in the sHLA-G levels between anti-glycoprotein (GP) IIbIIIa and anti-GPIb-IX autoantibodies groups. Interestingly, sHLA-G is positively correlated with platelet counts in ITP patients both with and without autoantibodies, suggesting that HLA-G can also protect platelets from destruction through other mechanisms. To elucidate this, we then analyzed both expression of membrane HLA-G and ILTs on PBMCs. Our data found less membrane-bound HLA-G and ILTs on CD4+T cells and CD14+cells in ITP patients. Moreover, recombinant human HLA-G (rhHLA-G) upregulated expression of ILT2 on CD4+and ILT4 on CD14+cells. The upregulation of ILT by rhHLA-G indicates that sHLA-G might also benefit ITP patients negative for anti-platelet autoantibodies.To determine whether rhHLA-G affects T cell differentiation, we tested 17 cytokines by ELISA to investigate the immune environment with or without rhHLA-G, and found that rhHLA-G upregulated Interleukin (IL)-4 and IL-10, and downregulated tumor necrosis factor-α, IL-12, and IL-17 secreted by ITP patient peripheral blood mononuclear cells (PBMCs), indicating a promotion of helper T(Th)2 and inhibition of Th1 and Th17 differentiation. However, our results indicated that rhHLA-G did not expand the CD4+CD25+Foxp3+regulatory T cells(Tregs). In addition, rhHLA-G exerted their immunosuppressive effects by upregulating IL-1β, IL-2, Granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). By reprogramming the cytokine profile in ITP patients, HLA-G established an optimal environment for the impaired cell populations, such as dendritic cells (DCs), to regain their tolerogenic function. Our results showed that rhHLA-G inhibited monocyte-derived DCs maturation by downregulating CD80 and CD86 expression. To further assess whether the rhHLA-G modulated-DCs were functionally tolerogenic, PBMCs were then cocultured with rhHLA-G modulated-DCs. Our results showed that rhHLA-G-modulated DCs from ITP patients suppressed CD4+T cell proliferation by 38.8% compared to unmodulated cells. Besides, PBMCs exposed to rhHLA-G were less cytotoxic toward plateletsfrom ITP patients or healthy subjects. This study provides a potential strategy to protect exogenous platelets from destruction in ITP patients.In conclusion, our data demonstrated that impaired expression of HLA-G and/or ILT is involved in the pathogenesis of ITP, and recombinant human HLA-G can restore immune tolerance in ITP patients via upregulation of ILTs, which indicates that HLA-G can be a diagnostic marker and also a therapeutic option for ITP treatment. DisclosuresNo relevant conflicts of interest to declare.

Novel molecules mediate specialized functions of human regulatory macrophages.

Now that adoptive transfer of regulatory macrophages (Mregs) is clinically practicable, we ask whether this approach could be used to achieve self-sustaining peripheral regulation and what mechanisms may be involved. Dehydrogenase/reductase 9 (DHRS9)-expressing Mregs are a specialized subset of monocyte-derived macrophages that are currently being investigated as a tolerogenic cell-based therapy. Human Mregs are defined by their capacity to convert naïve CD4 T cells to IL-10-secreting FoxP3 regulatory T cells (Tregs) through an activation-dependent process involving signals mediated by TGF-β, retinoic acid, indoleamine 2,3-dioxygenase activity, notch and progestagen associated endometrial protein (PAEP). Mreg-induced iTregs (miTregs) are a phenotypically distinct type of in-vitro-derived human iTreg that expresses butyrophilin-like protein 8 (BTNL8) and T cell immunoreceptor with Ig and ITIM domains (TIGIT). miTregs are nonspecifically suppressive of mitogen-stimulated bystander T cell proliferation and inhibit TNFα-induced maturation of monocyte-derived dendritic cells. Preclinical and clinical studies find that intravenous infusion of allogeneic Mregs leads to enrichment of circulating TIGIT Tregs. These results suggest a feed-forward mechanism by which Mreg treatment could promote solid organ transplant acceptance through rapid induction of direct pathway Tregs.

Pancreatic-cancer-cell-derived trefoil factor 2 impairs maturation and migration of human monocyte-derived dendritic cells in vitro

ABSTRACTPancreatic cancer is a challenging disease with a high mortality rate. While the importance of crosstalk between cancer and immune cells has been well documented, the understanding of this complex molecular network is incomplete. Thus, identification of the secreted proteins contributing to the immunosuppressive microenvironment in pancreatic cancer is crucial for effective diagnosis and/or therapy. We utilized a public microarray dataset (GSE16515) from the Gene Expression Omnibus database to identify genes for secreted proteins in pancreatic cancer. RT–PCR and ELISA of the pancreatic cancer cell lines validated the cellular origin of the selected genes. For functional assay of the selected proteins, we utilized human-monocyte-derived dendritic cells (DCs). From the list of the secreted proteins, trefoil factor 2 (TFF2) was further examined as a potential chemokine/cytokine. While TFF2 did not significantly affect the phenotypic maturation and the allostimulatory capacity of DCs, TFF2 preferentially attracted immature (but not mature) DCs and inhibited their endocytic activity. Our data suggest that TFF2 from pancreatic cancer cells may attract immature DCs and affect the initial stage of DC maturation, thereby contributing to the induction of immune tolerance against pancreatic cancer.

Automated closed-system manufacturing of human monocyte-derived dendritic cells for cancer immunotherapy

Dendritic cell (DC)-based vaccines have been successfully used for immunotherapy of cancer and infections. A major obstacle is the need for high-level class A cleanroom cGMP facilities for DC generation. The CliniMACS Prodigy® (Prodigy) represents a new platform integrating all GMP-compliant manufacturing steps in a closed system for automated production of various cellular products, notably T cells, NK cells and CD34+ cells. We now systematically tested its suitability for producing human mature monocyte-derived DCs (Mo-DCs), and optimized it by directly comparing the Prodigy approach to our established standard production of Mo-DCs from elutriated monocytes in dishes or bags. Upon step-by-step identification of an optimal cell concentration for the Prodigy's CentriCult culture chamber, the total yield (% of input CD14+ monocytes), phenotype, and functionality of mature Mo-DCs were equivalent to those generated by the standard protocol. Technician's labor time was comparable for both methods, but the Prodigy approach significantly reduced hands-on time and high-level clean room resources. In summary, using our optimized conditions for the CliniMACS Prodigy, human Mo-DCs for clinical application can be generated almost automatically in a fully closed system. A significant drawback of the Prodigy approach was, however, that due to the limited size of the CentriCult culture chamber, in contrast to our standard semi-closed elutriation approach, only one fourth of an apheresis could be processed at once.

Tobacco smoke and nicotine suppress expression of activating signaling molecules in human dendritic cells

Cigarette smoke has significant toxic effects on the immune system, and increases the risk of developing autoimmune diseases; one immunosuppressive effect of cigarette smoke is that it inhibits the T cell-stimulating, immunogenic properties of myeloid dendritic cells (DCs). As the functions of DCs are regulated by intra-cellular signaling pathways, we investigated the effects of cigarette smoke extract (CSE) and nicotine on multiple signaling molecules and other regulatory proteins in human DCs to elucidate the molecular basis of the inhibition of DC maturation and function by CSE and nicotine. Maturation of monocyte-derived DCs was induced with the TLR3-agonist poly I:C or with the TLR4-agonist lipopolysaccharide, in the absence or presence of CSE or nicotine. Reverse-phase protein microarray was used to quantify multiple signaling molecules and other proteins in cell lysates. Particularly in poly I:C-matured DCs, cigarette smoke constituents and nicotine suppressed the expression of signaling molecules associated with DC maturation and T cell stimulation, cell survival and cell migration. In conclusion, constituents of tobacco smoke suppress the immunogenic potential of DCs at the signaling pathway level.

Type I and III Interferon Productions Are Impaired in X-Linked Agammaglobulinemia Patients Toward Poliovirus but Not Influenza Virus.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) mutation. Patients are susceptible to severe enterovirus infections. The underlying mechanism remains unknown. BTK is involved in toll-like receptors pathway, which initiates antiviral responses including interferon (IFN) productions. To demonstrate type I and III IFN productions in dendritic cells of XLA patients is decreased in response to oral poliovirus vaccine (OPV) but not H1N1 virus. Monocyte-derived dendritic cells (MoDCs) were derived from nine XLA patients aged 22-32 years old and 23 buffy coats from Hong Kong Red Cross blood donors. LFM-A13 was used to inhibit BTK. OPV Sabin type 1 and H1N1 influenza virus were used to stimulate MoDCs with RPMI as mock stimulation. The antiviral cytokine productions and phenotypic maturation of MoDCs were determined 24 h post-stimulation. OPV RNA was determined at 0, 6, 12, and 24 h post-stimulation. Upon OPV stimulation, IFN-α2, IFN-β, and IFN-λ1 productions in MoDCs from XLA patients and BTK-inhibited MoDCs of healthy controls were significantly lower than that from healthy controls. Whereas upon H1N1 stimulation, the IFN-α2, IFN-β, and IFN-λ1 productions were similar in MoDCs from XLA patients, BTK-inhibited MoDCs of healthy controls and healthy controls. The mean fluorescent intensities (MFI) of CD83, CD86, and MHC-II in MoDCs from XLA patients in response to OPV was similar to that in response to mock stimulation, while the MFI of CD83, CD86, and MHC-II were significantly higher in response to H1N1 stimulation than that in response to mock stimulation. Whereas, the MFI of CD83, CD86, and MHC-II in MoDCs of healthy controls were significantly higher in response to both OPV and H1N1 stimulation compared to that in response to mock stimulation. Production of type I and III IFN in response to OPV was deficient in MoDCs from XLA patients, but was normal in response to H1N1 due to deficient BTK function. Moreover, phenotypic maturation of MoDCs from XLA patients was impaired in response to OPV but not to H1N1. These selective impairments may account for the unique susceptibility of XLA patients toward severe enterovirus infections.