scholarly journals Phosphate-modified CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells

2020 ◽  
Vol 24 (6) ◽  
pp. 653-660
Author(s):  
A. A. Ostanin ◽  
O. Y. Leplina ◽  
E. A. Burakova ◽  
T. V. Tyrinova ◽  
A. A. Fokina ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cpg Odn ◽  
Blood Monocytes ◽  
Myeloid Dendritic Cells ◽  
Gm Csf ◽  
Cpg Oligodeoxynucleotides ◽  
Cpg Oligonucleotides ◽  
Cpg Odns ◽  
Dc Maturation

Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D-SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.

scholarly journals In-VitroDifferentiation of Mature Dendritic Cells From Human Blood Monocytes

1998 ◽  
Vol 6 (1-2) ◽  
pp. 25-39 ◽  
Author(s):  
Robert Gieseler ◽  
Dirk Heise ◽  
Afsaneh Soruri ◽  
Peter Schwartz ◽  
J. Hinrich Peters
Keyword(s):  
Dendritic Cells ◽  
Human Blood ◽  
T Cell Response ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Hla Dr ◽  
Hla Dq ◽  
Specific Stimulation ◽  
Antigen Presenting

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-γto differentiate monocyte-derived DCin vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class I++/+++HLA-DR++/+++HLA-DP+, and HLA-DQ+. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD 11c+CD45R0+blood DC subset identified earlier, their differentiation in the presence of the Thl and Th2 cytokines IFN-γand IL-4 indicates that these DC may conform to mature mucosal DC.

Leukemic blasts fail to differentiate into mature antigen presenting cells, and may hinder dendritic cell maturation in vitro and in vivo

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 10556-10556
Author(s):  
J. Rosenblatt ◽  
R. Stone ◽  
C. Lenahan ◽  
Z. Wu ◽  
B. Vasir ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Tumor Vaccine ◽  
Leukemia Cells ◽  
Phenotypic Characteristics ◽  
Gm Csf ◽  
Maturation In Vitro ◽  
Dc Maturation

10556 Background: Dendritic cells (DC) play a key role in the development of tumor specific immune responses. Dendritic cells differentiated from leukemic blasts (LDC) are being explored as a tumor vaccine in AML. We examined the phenotypic and functional characteristics of LDC, the phenotypic characteristics of native DC in AML patients, and the effect of leukemic blasts on the phenotype of DC generated from normal donors. Methods: Leukemia blasts were isolated from peripheral blood of 24 patients with AML. LDC were generated by culturing blasts in the presence of GM-CSF, IL-4 and TNFa for 7 days. The phenotype of circulating DC1 (CD11C+/lin-) and DC2 (CD123+/ lin-) in AML patients was assessed by multichannel FACS analysis. To assess the effect of blasts on DC maturation, adherent mononuclear cells were isolated from normal donors, combined with leukemia cells in a 10:1 ratio, and cultured with GM-CSF, IL-4, and TNFa. Results: LDC demonstrate only modest expression of the costimulatory molecules CD80 and CD86 (mean expression 10% and 32%) and poorly express the maturation marker CD83 (mean expression 4%). Interferon gamma production by autologous T cells was not higher after stimulation with LDC than with blasts. LDC stimlation resulted in a 2 fold increase in both CD4+/CD25+/CD69+ (activated) and CD4+/CD25+/FOXP3+ (regulatory) T cells. Given the inability of leukemia progenitors to differentiate into phenotypically mature DC, we assessed whether leukemia cells directly inhibit differentiation of DC from normal progenitors. Expression of costimulatory molecules was decreased in DC differentiated in the presence of blasts. Mean expression of CD80, CD83, and CD86 was 16%, 2%, 83% and 49%, 10%, 99% for DCs generated in the presence or absence blasts respectively. Phenotypic characteristics of native DC in patients with AML were examined. In 3 experiments, a predominance of DC2 was seen (ratio DC2/DC1 5), and both DC1 and DC2 poorly expressed CD83 (mean expression 9% DC1, 0.9% DC2). Conclusions: LDC have phenotypic and functional deficiencies, limiting their efficacy as a tumor vaccine. Contact with leukemic blasts may inhibit DC maturation in vitro and in vivo, which may contribute to the lack of effective antitumor immunity in AML patients. No significant financial relationships to disclose.

scholarly journals Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Ludmila V. Sakhno ◽  
Ekaterina Ya. Shevela ◽  
Marina A. Tikhonova ◽  
Sergey D. Nikonov ◽  
Alexandr A. Ostanin ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Pulmonary Tuberculosis ◽  
Peripheral Blood ◽  
Antigen Presenting Cells ◽  
T Cell Response ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Gm Csf ◽  
Antigen Presenting

The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generatedin vitromacrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity toM. tuberculosisantigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14+CD16+expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which werein vitrogenerated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γcoupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generatedin vitrofrom peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γproduction in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response toM. tuberculosiswas discussed.

scholarly journals Monomethyl fumarate treatment impairs maturation of human myeloid dendritic cells and their ability to activate T cells

2017 ◽  
Vol 25 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Maria Antonietta Mazzola ◽  
Radhika Raheja ◽  
Keren Regev ◽  
Vanessa Beynon ◽  
Felipe von Glehn ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Dimethyl Fumarate ◽  
Nuclear Factor Κb ◽  
Interleukin 17 ◽  
Myeloid Dendritic Cells ◽  
Gm Csf ◽  
Monomethyl Fumarate ◽  
And Function

Background: Dimethyl fumarate (DMF) and its active metabolite monomethyl fumarate (MMF) effectively lead to reduction in disease relapses and active magnetic resonance imaging (MRI) lesions. DMF and MMF are known to be effective in modulating T- and B-cell responses; however, their effect on the phenotype and function of human myeloid dendritic cells (mDCs) is not fully understood. Objective: To investigate the role of MMF on human mDCs maturation and function. Methods: mDCs from healthy controls were isolated and cultured in vitro with MMF. The effect of MMF on mDC gene expression was determined by polymerase chain reaction (PCR) array after in vitro MMF treatment. The ability of mDCs to activate T cells was assessed by in vitro co-culture system. mDCs from DMF-treated multiple sclerosis (MS) patients were analyzed by flow cytometry and PCR. Results: MMF treatment induced a less mature phenotype of mDCs with reduced expression of major histocompatibility complex class II (MHC-II), co-stimulatory molecules CD86, CD40, CD83, and expression of nuclear factor κB (NF-κB) subunits RELA and RELB. mDCs from DMF-treated MS patients also showed the same immature phenotype. T cells co-cultured with MMF-treated mDCs showed reduced proliferation with decreased production of interferon gamma (IFN-γ), interleukin-17 (IL-17), and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to untreated cells. Conclusion: We report that MMF can modulate immune response by affecting human mDC function.

8. Examining the Effects of Peptide-Gold Nanoparticle Hybrids on the Activation of Myeloid Dendritic Cells

2016 ◽  
Author(s):  
Annie Liu
Keyword(s):  
Dendritic Cells ◽  
Immune Modulation ◽  
Immune Therapy ◽  
Myeloid Dendritic Cells ◽  
Gm Csf ◽  
Enhanced Expression ◽  
Microscope Imaging ◽  
Macrophage Colony ◽  
And Function

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in innate and adaptive immunity. They have become a major target for immune modulation based therapeutic strategies. Gold nanoparticles (GNPs) are biocompatible materials. The surface of GNPs can be easily modified with peptides via thiol-gold linkage to alter their surface chemistry and to modulate their cellular uptake. The purpose of this present study was to investigate the ability of peptide-GNP hybrids to modulate the DC immune response in vitro. To assess the activation effects of peptide-GNP hybrids, DCs were differentiated from bone marrow (BM) cells of black B57BL/6 mice by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At day 9 of culture, BM-derived DCs (BMDCs) were exposed to GNP hybrids with a diameter of approximately 10 nm. The purity of the resulting DCs was determined by the CD11c positive cell population measured by Flow Cytometry. We found that the peptide-GNP hybrids were internalized by immature BMDC via bright field microscope imaging. Furthermore, the addition of GNPs to immature BMDCs was found to influence both phenotype and function of immature BMDCs. Stimulation by peptide-GNP hybrids on the immature BMDCs led to an enhanced expression of co-stimulatory molecules on the cell surface, including CD40, CD80 and CD86, and a secretion of cytokines in the culture medium. This observation suggests that peptide-GNP stimulation can lead to the activation of BMDCs, and implies a potential application for strengthening vaccine delivery devices or cancer immune therapy.

Serial Analysis of Gene Expression in Human Monocyte-Derived Dendritic Cells

Blood ◽  
1999 ◽  
Vol 94 (3) ◽  
pp. 845-852 ◽  
Author(s):  
Shin-ichi Hashimoto ◽  
Takuji Suzuki ◽  
Hong-Yan Dong ◽  
Shigenori Nagai ◽  
Nobuyuki Yamazaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Cell Structure ◽  
Human Monocyte ◽  
Interleukin 4 ◽  
Maturation Stage ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Highly Expressed Genes

Dendritic cells (DCs) are professional antigen-presenting cells in the immune system and can be generated in vitro from hematopoietic progenitor cells in the bone marrow, CD34+ cord blood cells, precursor cells in the peripheral blood, and blood monocytes by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4, and tumor necrosis factor-. We have performed serial analysis of gene expression (SAGE) in DCs derived from human blood monocytes. A total of 58,540 tag sequences from a DC complementary DNA (cDNA) library represented more than 17,000 different genes, and these data were compared with SAGE analysis of tags from monocytes (Mo) and GM-CSF–induced macrophages (M◊). Many of the genes that were differentially expressed in DCs were identified as genes encoding proteins related to cell structure and cell motility. Interestingly, the highly expressed genes in DCs encode chemokines such as TARC, MDC, and MCP-4, which preferentially chemoattract Th2-type lymphocytes. Although DCs have been considered to be very heterogeneous, the identification of specific genes expressed in human Mo-derived DCs should provide candidate genes to define subsets of, the function of, and the maturation stage of DCs and possibly also to diagnose diseases in which DCs play a significant role, such as autoimmune diseases and neoplasms. This study represents the first extensive gene expression analysis in any type of DCs.

scholarly journals TARM1 contributes to development of arthritis by activating dendritic cells through recognition of collagens

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rikio Yabe ◽  
Soo-Hyun Chung ◽  
Masanori A. Murayama ◽  
Sachiko Kubo ◽  
Kenji Shimizu ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Bone Marrow Cells ◽  
Dendritic Cell Maturation ◽  
Mouse Bone Marrow ◽  
Gm Csf ◽  
Good Target ◽  
Antigen Presenting ◽  
Dc Maturation

AbstractTARM1 is a member of the leukocyte immunoglobulin-like receptor family and stimulates macrophages and neutrophils in vitro by associating with FcRγ. However, the function of this molecule in the regulation of the immune system is unclear. Here, we show that Tarm1 expression is elevated in the joints of rheumatoid arthritis mouse models, and the development of collagen-induced arthritis (CIA) is suppressed in Tarm1–/– mice. T cell priming against type 2 collagen is suppressed in Tarm1–/– mice and antigen-presenting ability of GM-CSF-induced dendritic cells (GM-DCs) from Tarm1–/– mouse bone marrow cells is impaired. We show that type 2 collagen is a functional ligand for TARM1 on GM-DCs and promotes DC maturation. Furthermore, soluble TARM1-Fc and TARM1-Flag inhibit DC maturation and administration of TARM1-Fc blocks the progression of CIA in mice. These results indicate that TARM1 is an important stimulating factor of dendritic cell maturation and could be a good target for the treatment of autoimmune diseases.

Receptor-type protein tyrosine phosphatase gamma (PTPγ), a new identifier for myeloid dendritic cells and specialized macrophages

Blood ◽  
2006 ◽  
Vol 108 (13) ◽  
pp. 4223-4231 ◽  
Author(s):  
Daniele Lissandrini ◽  
William Vermi ◽  
Marzia Vezzalini ◽  
Silvano Sozzani ◽  
Fabio Facchetti ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Protein Tyrosine Phosphatase ◽  
Human Peripheral Blood ◽  
Tyrosine Phosphatase ◽  
Advanced Pancreatic Cancer ◽  
Lymphoid Tissues ◽  
Blood Monocytes ◽  
Myeloid Dendritic Cells ◽  
Protein Tyrosine

AbstractProtein tyrosine phosphatase (PTPγ) is a receptor-like molecule with a known role in murine hematopoiesis. We analyzed the regulation of PTPγ expression in the human hematopoietic system, where it was detected in human peripheral blood monocytes and dendritic cells (DCs) of myeloid and plasmacytoid phenotypes. Its expression was maintained during in vitro monocyte differentiation to dendritic cells (moDC) and was further increased after maturation with bacterial lipopolysaccharide (LPS), CD40L, and TNFα. But PTPγ was absent when monocytes from the same donor were induced to differentiate in macrophages. B and T lymphocytes did not express PTPγ. Rather, PTPγ mRNA was expressed in primary and secondary lymphoid tissues, and the highest expression was in the spleen. PTPγ was detected by immunohistochemistry in subsets of myeloid-derived DCs and specialized macrophages (tingible bodies, sinus and alveolar macrophages). Classic macrophages in infective or reactive granulomatous reactions did not express PTPγ. Increased PTPγ expression was associated with a decreased ability to induce proliferation and interferon-γ secretion in T cells by moDCs from patients with advanced pancreatic cancer. Taken together, these results indicate that PTPγ is a finely regulated protein in DC and macrophage subsets in vitro and in vivo.

Endothelial Cell-Derived Microparticules (EMPs) Can Induce In Vitro Maturation of Both Plasmocytoid and Myeloid Dendritic Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1273-1273
Author(s):  
Francine Garnache-Ottou ◽  
Fanny Angelot ◽  
Sabeha Biichle ◽  
Joel PLumas ◽  
Francoise Dignat George ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Endothelial Cell ◽  
Cell Lineage ◽  
Annexin V ◽  
Negative Control ◽  
Dose Effect ◽  
Blood Monocytes ◽  
Myeloid Dendritic Cells ◽  
Luminex Technology

Abstract The implication of plasmocytoid dendritic cells (pDCs) in transplantation is more and more reported, especially because of their tolerogenic properties supported by the expansion of regulatory T cells. Endothelial microparticles (EMPs) are plasma membrane-derived vesicules shed from endothelial stimulation involved in physiological and pathological processes, including intercellular communication, hemostasis and immunity. However, the interaction between DC and EMP are not well-known, especially in the context of alloreactivity. Therefore, the aims of the current study were to explore the DC/EMP interactions, more specifically the capacity of EMPs to modulate the maturation of DC. Firstly, we validated an original co-culture model involving a tumoral pDC lineage (GEN 2.2) and EMPs generated in a standard fashion from an endothelial cell lineage (HMEC U608), stimulated or not with TNF-α . EMPs was analyzed by FACS (FITC labelled annexin V) and supernatant resulting from the last wash of EMPs was used as negative control. By incubating increasing amounts of EMPs we showed that EMPs can lead to GEN pDC maturation in a dose- effect manner as evidenced by a significative increase in the HLA-DR and CD83 expression, compared to the negative controls or, of importance, compared to the maturation with CPG-A (ligand of TLR9 mimicking bacterial DNA), CD40L or R848 (ligand of TLR7 mimicking viral RNA). To confirm and expand these findings, we then cocultured with EMPs (and similar controls) circulating pDC obtained by an anti-BDCA-4 MoAb-mediated positive selection of healthy donors PBMC , and myeloid DC (mDC) obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4. Maturation of both normal circulating pDC and mDC occurred in the presence of EMPs and once again, in a dose-dependant fashion. The cytokine secretion pattern of the EMPs- matured mDC and pDC, determined by the Luminex technology, was as expected for such cell populations. The EMPs matured pDC secreted IL-1, IL-6, IFN-α , IL-12 and IL-10 but not TGF-β . In contrast, the EMPs matured mDC secreted mainly proinflammatory cytokines : IL-1, IL-6 and IL-8 but not IL-10. Overall, our studies demonstrate that EMPs can induce the maturation of both pDC and mDC. Such endothelial microparticules, generated by inflammatory and hemostatic disorders, could play a role in post-transplant immune responses. Determining the factors resulting in EMPs-induced pDC vs mDC maturation may open the possibility for EMPs-related immuno-intervention.

Generation of dendritic cells using viral lysate of tumor cells in vitro.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15202-e15202
Author(s):  
Sergey A. Kolpakov ◽  
Elena P. Kolpakova ◽  
Anastasia O. Sitkovskaya ◽  
Elena Yu. Zlatnik ◽  
Svetlana Yu. Filippova ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cytopathic Effect ◽  
Cytotoxic Lymphocytes ◽  
Blood Monocytes ◽  
U87mg Cell ◽  
Membrane Expression ◽  
Gm Csf ◽  
Highly Active ◽  
Hla Dr

e15202 Background: The purpose of the study was to investigate the effect of a new unclassified rotavirus on the effectiveness of maturation of dendritic cells (DC) and their activation of lymphocytes in vitro. Methods: Strain No.100 of the RVK virus was isolated by S.A. Kolpakov and characterized as rotavirus. U87MG cell lysate (glioblastoma) was obtained by incubation with 108 RVK particles in the DMEM medium with L-glutamine for 72 hours; a cytopathic effect was observed. Immature DCs were cultured for 7 days in the presence of IL-4 and GM-CSF. For antigen loading of DCs, we used the following options for 48 h: a) U87MG lysate obtained by repeated freeze-thaw cycles (TL); b) U87MG lysate obtained by co-cultivation with RVK (VTL); c) RVK. To assess the DC ability to activate autologous lymphocytes, they were co-cultured for 5 days in a 3:1 ratio. Results: The use of VTL culture for DC loading caused an increase in the expression of mature DC (mDC) markers compared to TL: the number of CD83+/CD86+ cells increased by more than 2 times, CD83+/CD80+ by 1.2 times, CD83+HLA-DR - by 5.5 times. With VTL, expression of markers of immature DCs (CD1a, CD14) was minimal. The use of RVK as an antigen induced the generation of DCs from monocytes, but their maturation was much less pronounced: a significant increase in the membrane expression of CD86, but not CD83, was determined. These DCs demonstrated a higher expression of markers of immature DCs, compared to stimulation with cell lysates (both TL and VTL): CD1a and CD14; CD80+/CD86+ level was the highest among all options. Analysis of the DC effect on co-cultivated lymphocytes showed that DCs loaded with RVK, both alone and as part of VTL, stimulated predominantly the NKT subpopulation. The same samples contained more T lymphocytes, activated CD4+ and CD8+ compared to samples stimulated by TL. However, the samples co-cultivated with VTL contained the maximal amount of CD4+/CD25+/CD127dim phenotypically corresponding to Tregs. Conclusions: The antigen loading of immature DCs with RVK alone causes their activation, but not maturation, which is not realized in typical terms of the DC generation from blood monocytes. The presence of RVK, including in VTL, has a stimulating effect on NKT lymphocytes suggesting the possible generation of specific highly active cytotoxic lymphocytes.

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