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.

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.

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.

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 POS0673 TOLEROGENIC DENDRITIC CELLS IN RHEUMATOID ARTHRITIS PATIENTS: NEWS AND PROMISES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 581.2-581
Author(s):  
Y. Kurochkina ◽  
E. Chernykh ◽  
A. Sizikov
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Dendritic Cells ◽  
Mixed Lymphocyte Culture ◽  
Lymphocyte Culture ◽  
Moderate Activity ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Hla Dr ◽  
Enhanced Expression

Background:Dendritic cells (DCs) are known to contribute to the pathogenesis of rheumatoid arthritis (RA) through presentation of cartilage glycoprotein, production of proinflammatory cytokines and activation of Th1/Th17 responses. Along with stimulating activity, DCs may exhibit suppressive functions via capacity to induce T cell apoptosis/anergy and to generate regulatory T cells. Since these DCs have potential to control autoreactive T-lymphocytes, the enhancing of tolerogenic properties of DCs seems to be a new important strategy in treatment of RA. Dexamethasone is widely used in clinical practice and can be used as a tolerogenic substance. Therefore, the properties of DCs generated in presence of dexamethasone are of great clinical interests.Objectives:The aim of our study is to describe the properties of tolerogenic DCs, generated with dexamethasone in patients with RA and their influence on autologous T-cells.Methods:Sixty five patients with RA with high and moderate activity of disease were recruited in this study. All patients follow ACR/EULAR criteria (2010). All studies were performed after receiving informed consent. All patients received conventional synthetic DMARDs. DCs were generated from blood monocytes culturing for 5 days with GM-CSF and IFN-α in the presence dexamethasone (dexDCS), applied on third day. LPS as maturation stimuli was added on fourth day. The expression of CD14, CD83, HLA-DR, TLR-2 on the surface of DCs was measured by flow cytometry. The functions of DCs were evaluated by measuring cytokine production and DCs allostimulatory activity in mixed lymphocyte culture. Mature DCs generated in absence of dexamethasone used as control.Results:We revealed that dexDCs are characterized by enhanced expression of CD14+cells and decreased number of CD83+cells but percent of HLA-DR+cells were constant (about 85). DexDCs show high expression of TLR-2 is seen as tolerogenic molecule (75%vs51%, p=0.05 compared to control). DexDCs also have marked prominent increase of TNFα/IL-10 ratio in contrast to control (0.59 vs 1.8, p=0.03). DexDCs suppressed proliferation of allogenic T-cells (2005 vs 7980 cpm, p=0.0002). To assess the stability of the DC in the proinfflamatory micro-environment after assessing stimulatory activity dexDCs were then cultivated with LPS and allostimulatory activity were evaluated one more. The stimulation activity dexDCs after incubation with LPS were not increase (4692 vs 6053 cpm, p=0.7). Also earlier we showed possibility of dexDCs induse apoptosis of autologous T-cells, activation of CD4+IL10+Tr1 and possession of antigen-specific suppression.Conclusion:The data obtained indicate that dexDCs from RA patients have the main tolerogenic features and stable in inflammatory environment that proves their potential in the treatment of rheumatoid arthritis.Disclosure of Interests:None declared

scholarly journals Phenotypic and genetic evaluation of human monocyte-derived dendritic cells generated from whole blood for immunotherapy

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yousri M. Hussein ◽  
Doaa M. Hendawy ◽  
Abdalrahman N. Alghamdy ◽  
Nermin Raafat
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Surface Antigens ◽  
Interleukin 4 ◽  
Blood Monocytes ◽  
Hla Dr ◽  
Significant Difference

Abstract Background Dendritic cells (DCs) recognize different pathogens and cancer cells and activate the adaptive immune response. The generation of effective DC-based cancer vaccines depends on the appropriate differentiation of monocytes in vitro. This study aimed to standardize a protocol for the in vitro differentiation of human peripheral blood monocytes into immature DCs upon treatment with growth factors and generate monocyte-derived DCs (MoDCs). Peripheral blood mononuclear cells were separated from peripheral blood. After monocyte enrichment by plastic adhesion, monocytes were cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate immature DCs. The cells were examined by microscopy. Using flow cytometry, DCs were evaluated for the expression of the CD83 and HLA-DR surface antigens, for the uptake of fluorescein isothiocyanate conjugated dextran, and also for the expression of CD80 and CD86 mRNA. Results CD80 and CD86 genes expression was upregulated at day six and exhibited a significant difference (P < 0.05). DCs showed positive expression of the CD83 and HLA-DR surface antigens by flow cytometry and FITC-conjugated dextran uptake. Conclusion This study represents a preliminary trial to generate immature MoDCs in vitro from blood monocytes collected by the flask adherence method. It offers a panel of surface markers for DCs characterization and provides Immature DCs for experimental procedures after 6 incubation days.

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 Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab

Blood ◽  
2002 ◽  
Vol 100 (5) ◽  
pp. 1715-1720 ◽  
Author(s):  
Andrea G. S. Buggins ◽  
Ghulam J. Mufti ◽  
Jonathan Salisbury ◽  
Jane Codd ◽  
Nigel Westwood ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Peripheral Blood ◽  
Keyhole Limpet Hemocyanin ◽  
Interleukin 4 ◽  
Primary Immune Response ◽  
Soluble Antigen ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Hla Dr

CAMPATH antibodies recognize CD52, a phosphatidylinositol-linked membrane protein expressed by mature lymphocytes and monocytes. Since some antigen-presenting dendritic cells (DCs) differentiate from a monocytic progenitor, we investigated the expression of CD52 on dendritic cell subsets. Four-color staining for lineage markers (CD3, 14, 16, 19, 20, 34, and 56), HLA-DR, CD52, and CD123 or CD11c demonstrated that myeloid peripheral blood (PB) DCs, defined as lineage−HLA-DR+CD11c+, express CD52, while expression by CD123+ lymphoid DCs was variable. Depletion of CD52+ cells from normal PB strongly inhibited their stimulatory activity in an allogeneic mixed lymphocyte reaction and also reduced the primary autologous response to the potent neoantigen keyhole limpet hemocyanin. CD52 is thus expressed by a myeloid subset of PBDCs that is strongly allostimulatory and capable of initiating a primary immune response to soluble antigen. Administration of alemtuzumab, a humanized monoclonal antibody against CD52, to patients with lymphoproliferative disorders or as conditioning for hematopoietic stem cell transplantation resulted in a marked reduction in circulating lineage−HLA-DR+ DCs (mean 31-fold reduction,P = .043). Analysis of monocyte-derived DCs in vitro revealed a reduction in CD52 expression during culture in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, with complete loss following activation-induced maturation with lipopolysaccharide. In contrast to the findings in PB, epidermal and small-intestine DCs did not express CD52, suggesting either that transit from blood to epidermis and gut is associated with loss of CD52 or that DCs in these tissues originate from another population of cells.

scholarly journals Dendritic Cell Profile Induced bySchistosoma mansoniAntigen in Cutaneous Leishmaniasis Patients

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Diego Mota Lopes ◽  
Jamille Souza Fernandes ◽  
Thiago Marconi de Souza Cardoso ◽  
Aline Michele Barbosa Bafica ◽  
Sérgio Costa Oliveira ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cutaneous Leishmaniasis ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Gm Csf ◽  
Surface Molecules ◽  
Hla Dr ◽  
Immune Mediated ◽  
Cell Profile

The inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is associated with the pathogenesis of disease. Conversely, the immune response induced byS. mansoniantigens is able to prevent immune-mediated diseases. The aim of this study was to evaluate the potential of theS. mansoniSm29 antigen to change the profile of monocyte-derived dendritic cells (MoDCs) from subjects with cutaneous leishmaniasis (CL)in vitro. Monocytes derived from the peripheral blood mononuclear cells of twelve patients were cultured with GM-CSF and IL-4 for differentiation into dendritic cells and then stimulated with solubleLeishmaniaantigen (SLA) in the presence or absence of Sm29 antigen. The expression of surface molecules associated with maturation and activation (HLA-DR, CD40, CD83, CD80, and CD86), inflammation (IL-12, TNF), and downregulation (IL-10, IL-10R) was evaluated using flow cytometry. We observed that the frequencies of HLA-DR, CD83, CD80, and CD86 as well as of IL-10 and IL-10R on MoDCs were higher in cultures stimulated with Sm29, compared to the unstimulated cell cultures. Our results indicate that the Sm29 antigen is able to activate regulatory MoDCs in patients with cutaneous leishmaniasis. It might be useful to control the inflammatory process associated with this disease.

scholarly journals Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis.

1989 ◽  
Vol 170 (3) ◽  
pp. 865-875 ◽  
Author(s):  
J M Alvaro-Gracia ◽  
N J Zvaifler ◽  
G S Firestein
Keyword(s):  
Rheumatoid Arthritis ◽  
Class Ii ◽  
Tnf Alpha ◽  
Blood Monocytes ◽  
Ifn Gamma ◽  
Normal Peripheral Blood ◽  
Gm Csf ◽  
Hla Dr ◽  
Class Ii Mhc ◽  
Hla Dq

Granulocyte/macrophage CSF (GM-CSF) has recently been identified in rheumatoid arthritis (RA) synovial effusions. To study a potential role for GM-CSF and other cytokines on the induction of HLA-DR expression on monocytes and synovial macrophages, we analyzed the relative ability of recombinant human cytokines to induce the surface expression of class II MHC antigens on normal peripheral blood monocytes by FACS analysis. GM-CSF (800 U/ml) (mean fluorescence channel 2.54 +/- 0.33 times the control, p less than 0.001) and IFN-gamma (100 U/ml) (5.14 +/- 0.60, p less than 0.001) were the most potent inducers of HLA-DR. TNF-alpha and IL-4 also increased HLA-DR expression, although to a lesser degree [1.31 +/- 0.06 (p less than 0.02) and 1.20 +/- 0.03 (p less than 0.01), respectively]. IL-1 (40 U/ml), IL-2 (10 ng/ml), IL-3 (50 U/ml), IL-6 (100 U/ml), and CSF-1 (1,000 U/ml) did not affect surface HLA-DR density. GM-CSF also increased HLA-DR mRNA expression and surface HLA-DQ expression, but decreased CD14 (a monocyte/macrophage antigen) expression. The effect of GM-CSF on HLA-DR was not mediated by the generation of IFN-gamma in vitro because it was not blocked by anti-IFN-gamma mAb. GM-CSF was additive with IL-4 and low amounts (less than 3 U/ml) of IFN-gamma and synergistic with TNF-alpha. Because we have recently reported that supernatants of cultured RA synovial cells produce a non-IFN-gamma factor that induces HLA-DR on monocytes, we then attempted to neutralize this factor with specific anti-GM-CSF mAb. Four separate synovial tissue supernatants were studied, and the antibody neutralized the HLA-DR-inducing factor in each (p less than 0.01).

scholarly journals Still Alive and Kicking: In-Vitro-Generated GM-CSF Dendritic Cells!

Immunity ◽  
2016 ◽  
Vol 44 (1) ◽  
pp. 1-2 ◽  
Author(s):  
Manfred B. Lutz ◽  
Kayo Inaba ◽  
Gerold Schuler ◽  
Nikolaus Romani
Keyword(s):  
Dendritic Cells ◽  
Gm Csf
Sign in / Sign up

Export Citation Format

Share Document