Isolation of Phagosomes from Dendritic Cells by Using Magnetic Beads

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (14) ◽  
Author(s):  
Bénédicte Manoury
Keyword(s):  
Dendritic Cells ◽  
Magnetic Beads

scholarly journals AB0062 CHARACTERIZATION OF IL-12 AND IL-23 REDUCTION BY TOFACITINIB IN MDCS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1332.2-1333
Author(s):  
N. Vincken ◽  
C. Angiolilli ◽  
S. Cardoso ◽  
A. Lopes ◽  
M. Olde-Nordkamp ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Psoriatic Arthritis ◽  
Mrna Expression ◽  
Intracellular Signaling ◽  
Magnetic Beads ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Myeloid Dendritic Cells ◽  
Ifn Γ

Background:Psoriatic arthritis (PsA) is a chronic inflammatory auto-immune disease characterized by an excessive production of pathogenic mediators that cause inflammation of the skin, peripheral joints, entheses and the spine. Among these, interleukin (IL)-23, IL-12, the IL-17 family and TNF constitute key players in PsA pathogenesis.1,2IL-23, consisting of IL23A (IL-23p19) and IL12B (IL-12p40) subunits, is predominantly produced by myeloid dendritic cells (mDCs). While the p19 subunit is unique to IL-23, the p40 subunit is shared with IL-12. Together, IL-12 and IL-23 play a crucial role in promoting the differentiation of naïve T lymphocytes into T helper (Th) interferon (IFN)-γ-producing Th1 or IL17-producing Th17 cells, respectively.3Small-molecule inhibitors, such as the JAK/STAT inhibitor Tofacitinib, have recently shown promising therapeutic potential in PsA clinical trials.4The inhibition of JAKs by Tofacitinib results in the direct suppression of multiple intracellular signaling pathways which constitute key hubs in the cytokine network.5However, whether Tofacitinib is able directly target IL-12/IL-23 production by mDCs has not yet been documented. Suppression of these canonical inflammatory pathways would provide further evidence that Tofacitinib is an effective drug in halting both innate and adaptive immune responses.Objectives:To evaluate the transcriptional and molecular events underlining IL-12 and IL-23 regulation by Tofacitinib in mDCs.Methods:Peripheral blood mononuclear cells from healthy donors were isolated by Ficoll gradient. Monocytes and myeloid dendritic cells (mDCs) were isolated by using magnetic beads on autoMACS. Monocytes were cultured for 6 days in the presence of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate monocyte-derived dendritic cells (moDCs). MoDCs were harvested, washed and put to rest for 1 day prior to stimulation, while mDCs were stimulated on the same day of isolation. Both moDCs and mDCs were pre-treated with Tofacitinib and then stimulated with either lipopolysaccharide (LPS) or combination of LPS with IFN-γ for 4 hours. Cytokines were measured using enzyme-linked immunosorbent assay (ELISA) and gene expression was assessed using quantitative polymerase chain reaction (qPCR).Results:Treatment of both mDCs and moDCs with Tofacitinib led to a decreased mRNA expression of IL-12 p40 (IL12B) in the presence of TLR4 and IFNγ co-stimulation. The decreasedIL12BmRNA expression also resulted in lower production of IL-12 p40 and IL-23 proteins in mDCs.Conclusion:In this work, we demonstrated for the first time that Tofacitinib can suppress the production of IL-23/IL-12 p40 subunit in mDCs, upon the condition that an active type II IFN signalling is also present in these cells. This observation indicates that specific factors, such as endogenous IFN-γ levels in the serum of PsA patients, can possibly predict differential responses to Tofacitinib treatment.References:[1]Gaffen SL. et al. The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat Rev Immunol. 2014 Sep;14(9):585-600[2]Bravo A, Kavanaugh A. Bedside to bench: defining the immunopathogenesis of psoriatic arthritis. Nat Rev Rheumatol. 2019 Nov;15(11):645-656[3]Floss DM. et al. Insights into IL-23 biology: From structure to function. Cytokine Growth Factor Rev. 2015 Oct;26(5):569-78[4]Berekmeri A. et al. Tofacitinib for the treatment of psoriasis and psoriatic arthritis. Expert Rev Clin Immunol. 2018 Sep;14(9):719-730[5]T Virtanen A. et al. Selective JAKinibs: Prospects in Inflammatory and Autoimmune Diseases. BioDrugs. 2019 Feb;33(1):15-32Disclosure of Interests:None declared

Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2808-2814 ◽  
Author(s):  
Zlatko Dembic ◽  
John-Arne Røttingen ◽  
Jérôme Dellacasagrande ◽  
Karl Schenck ◽  
Bjarne Bogen
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Single Cell ◽  
Mhc Class Ii ◽  
Messenger Rna ◽  
Magnetic Beads ◽  
Specific Antigen ◽  
Class Ii ◽  
Single Cell Level ◽  
Cell Level

Abstract Antigen-presenting cells (APCs) from subcutaneous mouse MOPC315 plasmacytoma phagocytosed immunoglobulin G–coated magnetic beads, enabling efficient isolation within 2 hours by magnetic separation (APC-MB). Cell morphology was heterogeneous, with some of the cells having dendrites. The surface phenotype of purified tumor APCs-MB was CD11b+, CD11c+, CD40+, CD80+, CD86+, and MHC class II+. Tumor APCs-MB expressed messenger RNA for fractalkine and ABCD-1 chemokines, and for CC-type chemokine receptors CCR5 and CCR7, indicating the presence of mature dendritic cells (DCs). Visualized at a single cell level within 4 hours after disruption of the tumor, APCs-MB induced rapid Ca++ mobilization in MHC class II–restricted tumor idiotype (Id)–specific cloned CD4+ T cells. In long-term assays, tumor APCs-MB induced proliferation of naive T cells from Id-specific T-cell receptor transgenic mice. The results suggest that tumor APCs-MB represent a heterogeneous cell population that includes myeloid-derived DCs of various stages of maturation. A considerable fraction (≥ 15%) of DCs is spontaneously primed with tumor-specific antigen.

scholarly journals TLR-2 Signal Pathway Is Enhanced in Myeloproliferative Neoplasm - Related to the Increased Infammatory Cytokine and Pathogenesis?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5202-5202 ◽  
Author(s):  
Guanfang Shi ◽  
Maryna Yarotska ◽  
Hui Chen ◽  
Abdelmalek Cherif ◽  
Ching Wong ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Inflammatory Cytokines ◽  
Inflammatory Cytokine ◽  
Magnetic Beads ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Myeloproliferative Neoplasms ◽  
Clonal Evolution ◽  
Myeloproliferative Neoplasm ◽  
Stimulation Of

Abstract Introduction Myeloproliferative neoplasms (MPNs) are associated with constitutional symptoms and high levels of circulating inflammatory cytokines. The etiology of this increase in inflammatory cytokines could be secondary to Jak2 mutation resulting in enhanced transduction signaling of cytokines. Toll-like receptors (TLRs) represent another activating mechanism by which their expression on antigen presenting cells, such as dendritic cells (DCs) and macrophages, serve as key pattern recognition receptors (PRR) which play a central role in induction of innate and adaptive immune responses resulting in the signaling of inflammatory cytokines. TNFα is one of the inflammatory cytokine which is significantly raised in MPN patients. Recent evidence suggests that TNFα may facilitate clonal expansion of JAK2 V617F mutation positive cells which leads to hypothesis suggesting that chronic inflammation is involved in the pathogenesis and clonal evolution in Philadelphia-negative chronic MPNs. Therefore, we investigated the role of TLRs and related cytokines in MPNs and we found that only TLR-2 was significantly elevated in MPNs. We further used the TLR-2 ligand, Pam3CSK4, to study its effects on the production inflammatory cytokines. Materials & Methods TLR assay: TLR-2, 4, 7, 9 quantification was performed by TLR staining of 5×105 mononuclear cells which were incubated with 5 μg/mlof conjugated TLR-2, 4, 7, 9 antibody and assayed by flow cytometry. Monocyte-derived dendritic cells (mdDC) and Inflammatory Cytokines. mdDC were generated from isolated monocytes (anti-CD14-coated magnetic beads) then by incubation with IL-4 and GM-CSF for 4 days. Immature mdDC (1×104/well) were either left untreated or stimulated with Pam3CSK4, in 96 well plates. After 48h, cells were incubated with 20% human AB serum in PBS containing 0.1% BSA and 0.1% NaN3 for 20 min at 4¡C. Thereafter, cells were stained with fluorescence-labeled mAb specific to CD80, CD83, (BD Biosciences). Cytokine levels were determined in supernatants harvested after 48h using the MSD (Meso Scale Discovery) System. Results. Levels ofTLR-2 were significantly elevated in patients with MPNs especially in those with polycythemia vera and essential thrombocythemia, but only marginally elevated in myelofibrosis (MF) patients, which likely may be attributable to the relatively lack in number of MF patients (Fig 1). In all patient subsets, TLR 4, 7, 9 levels were not significantly different from controls as shown in Fig 1. Fig 2 shows maturation of mdDC by TLR-2 ligand, Pam3CSK4. Higher numbers of CD83+ cells were generated more from patients with elevated TLR levels, confirming these MPN patients has elevated TLR2 levels to be able to generated more dendric cells by TLR-2 ligand. Fig 3 a and b shows in elevated TLR-2 level MPN patients, Pam3CSK4 stimulated more cytokins of IFNγ, IL12p70, TNFα, IL6, IL8, IL10, IL13. Fig 4 showed in elevated TLR-2 levels MPN patients have more of IL8, TNFα, IL13, IL6, in the plasma, consistent with inflammatory cytokines likely from elevated elevated TLR-2.Therefore TLR-2 likely plays a very important role in the inflammatory cytokine production in MPN diseases. Conclusion. TLR-2 and not TLR-4, 7, 9 were found to be significantly elevated in MPNs. Stimulation of the TLR-2 ligand promoted the generation of more mature DCs in TLR-2 elevated MPN patients, demonstrating TLR-2 were elevated in these patients. Further, the stimulation of mdDC cultures with TLR2 ligand led to greater cytokine production and the level of plasma cytokines correlated with TLR-2 levels. We conclude that TLR-2 is important in the production of inflammatory cytokines in MPNs and it may play a significant role in the pathogenesis of MPNs. Disclosures Wang: Celgene: Research Funding.

GITR Ligand on Leukemic Myeloid Dendritic Cells Suppresses Induction of Leukemia-Associated Antigen-Specific CTLs from Naïve CD8+ T Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2347-2347
Author(s):  
Yukio Kondo ◽  
Takamasa Katagiri ◽  
Kinya Ohata ◽  
Shinji Nakao
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd8 T Cells ◽  
Magnetic Beads ◽  
Nk Cell ◽  
Leukemic Cells ◽  
Myeloid Dendritic Cells ◽  
Cancer Immunoediting ◽  
Specific Ctls ◽  
Anti Tumor Activity

Abstract Glucocorticoid-induced TNFR-related protein (GITR), a member of the TNF receptor superfamily, is expressed at low levels on resting T cells and is up-regulated following activation. Triggering of GITR with its ligand (GITRL) has been described to abrogate the function of CD4+CD25+ regulatory T cells and stimulate effector T cells in mice, but little is known about roles of GITR-GITRL interaction in humans. Recent evidence suggests that the interaction between GITR on NK cell and GITRL, which is constitutively expressed on tumor cells, negatively regulates NK cell-mediated anti-tumor activity in cancer patients. Leukemic myeloid dendritic cells (mDCs) can induce cyclin-dependent kinase (CDK2)- specific cytotoxic T lymphocytes (CTLs) from naïve T cells and CDK2-specific CTLs are detectable in MRD+ patients with leukemia after allo-SCT (Blood. 110 (11): a3228. 2007). The GITR-GITRL interaction may affect this process and a modification of the interaction may improve the efficiency of inducing CDK2-specific CTLs. Therefore, the expression of GITRL on leukemic cells and leukemic mDCs was examined to determine whether an engagement of GITR controls the priming of leukemia-associated-antigen (LAA)-specific CD8+ T cells. When cryopreserved BMMCs obtained at diagnosis from 5 patients with AML were assessed using flow cytometry, GITRL expression was detectable on leukemic cells in 3 patients. Leukemic mDCs were enriched from PBMCs of HLA-A24+ patients with anti-CD1c mAb-conjugated magnetic beads and were assessed for their ability to stimulate HLA-A24+ naïve CD8+ T cells to acquire cytotoxicity specific to CDK2-peptides (CDK2 158–166, CDK2 178–186). A three days culture of immature leukemic mDCs in the presence of TNFα up-regulated the expression of GITRL along with CD83 and CD40 expression. Naïve CD8+ T cells isolated from healthy individuals and cord blood were cultured with the GITRL-expressing leukemic mDCs in the presence or absence of anti- GITR monoclonal antibodies (mAbs) for 14 days and stained for CDK2 158–166/A24, CDK2 178–186/A24 pentamers. Blocking of GITR with the mAbs augmented induction of CDK2 158–166- and CDK2 178–186- specific CD8+ T cells from 0.37% to 1.17% and from 0.45% to 1.64%, respectively (Fig). Anti-GITR mAbs did not enhance induction of CDK2-specific T cells by peptide-pulsed monocyte-derived DCs which do not express GITRL. These data suggest that the expression of GITRL on circulating leukemic mDCs may suppress induction of CTLs specific to LAAs and induce cancer immunoediting in patients with leukemia. Administration of anti-GITR mAb after allo-SCT may enhance graft versus leukemia effect by CDK2-specific CTLs without vaccination of CDK2-peptides. Fig Blocking of GITR on T cells augments induction of CDK2-specific CTLs Fig. Blocking of GITR on T cells augments induction of CDK2-specific CTLs

scholarly journals Characterization of Murine Lung Dendritic Cells Infected with Mycobacterium tuberculosis

2001 ◽  
Vol 69 (2) ◽  
pp. 1127-1133 ◽  
Author(s):  
Mercedes Gonzalez-Juarrero ◽  
Ian M. Orme
Keyword(s):  
Dendritic Cells ◽  
Mycobacterium Tuberculosis ◽  
Magnetic Beads ◽  
Flow Cytometric Analysis ◽  
Interleukin 12 ◽  
Tissue Sections ◽  
Immature Dendritic Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Low Levels ◽  
Macrophage Colony

ABSTRACT Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-Ab+ and CD11c+ cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c+ and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8α antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytose Mycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naı̈ve and immune T cells to produce gamma interferon.

scholarly journals The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.

1992 ◽  
Vol 176 (1) ◽  
pp. 47-58 ◽  
Author(s):  
D Vremec ◽  
M Zorbas ◽  
R Scollay ◽  
D J Saunders ◽  
C F Ardavin ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Magnetic Beads ◽  
Cell Receptor ◽  
Class Ii ◽  
Immunofluorescent Staining ◽  
Alpha Chain ◽  
Class Ii Mhc ◽  
Antigen Presenting

A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.

Uptake of Magnetic Beads by Dendritic Cells, a Major Obstacle for the Use of Beads in Biological Assays

2004 ◽  
Vol 112 (3) ◽  
pp. 172-176 ◽  
Author(s):  
Petra Lütjens ◽  
Rüdiger Siekmeier ◽  
Veit Hölzel ◽  
Michael Bedorf ◽  
Peter Hanfland ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Magnetic Beads ◽  
Major Obstacle ◽  
Biological Assays

scholarly journals Generation of Immune Responses against Hepatitis C Virus by Dendritic Cells Containing NS5 Protein-Coated Microparticles

2008 ◽  
Vol 16 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Stephan Gehring ◽  
Stephen H. Gregory ◽  
Philip Wintermeyer ◽  
Costica Aloman ◽  
Jack R. Wands
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Responses ◽  
Magnetic Beads ◽  
Intracellular Cytokine Staining ◽  
Tyrosine Kinase Receptor ◽  
Cellular Immune Responses ◽  
Cellular Immune

ABSTRACT Dendritic cells (DCs) internalize and process antigens as well as activate cellular immune responses. The aim of this study was to determine the capacity of DCs that contain antigen-coated magnetic beads to induce immunity against the nonstructural hepatitis C virus (HCV) antigen 5 (NS5). Splenocytes derived from Fms-like tyrosine kinase receptor 3 (Flt3) ligand-pretreated BALB/c mice were incubated with magnetic beads coated with HCV NS5, lipopolysaccharide (LPS), and/or anti-CD40; purified; and used for immunization. Cellular immunity was measured using cytotoxic T-lymphocyte (CTL) and T-cell proliferation assays, intracellular cytokine staining, and a syngeneic tumor challenge using NS5-expressing SP2/0 myeloma cells in vivo. Splenocytes isolated from animals vaccinated with DCs containing beads coated with NS5, LPS, and anti-CD40 secreted elevated levels of interleukin-2 (IL-2) and gamma interferon in the presence of NS5. The numbers of CD4+, IL-2-producing cells were increased >5-fold in the group immunized with DCs containing beads coated with NS5, LPS, and anti-CD40, paralleled by an enhanced splenocyte proliferative response. Immunization promoted antigen-specific CTL activity threefold compared to the level for control mice and significantly reduced the growth of NS5-expressing tumor cells in vivo. Thus, strategies that employ NS5-coated beads induce cellular immune responses in mice, which correlate well with the natural immune responses that occur in individuals who resolve HCV.

Dendritic cells of the human epidermis: immuno-electron microscopic studies of la antigen reactivity

1978 ◽  
Vol 36 (2) ◽  
pp. 644-645
Author(s):  
G. Rowden ◽  
M. G. Lewis ◽  
T. M. Phillips
Keyword(s):  
Dendritic Cells ◽  
Langerhans Cells ◽  
Cell Types ◽  
Cell Type ◽  
Electron Microscopic ◽  
La Antigen ◽  
Microscopic Studies ◽  
A Cell ◽  
C3 Receptors ◽  
Antigen Reactivity

Langerhans cells of mammalian stratified squamous epithelial have proven to be an enigma since their discovery in 1868. These dendritic suprabasal cells have been considered as related to melanocytes either as effete cells, or as post divisional products. Although grafting experiments seemed to demonstrate the independence of the cell types, much confusion still exists. The presence in the epidermis of a cell type with morphological features seemingly shared by melanocytes and Langerhans cells has been especially troublesome. This so called "indeterminate", or " -dendritic cell" lacks both Langerhans cells granules and melanosomes, yet it is clearly not a keratinocyte. Suggestions have been made that it is related to either Langerhans cells or melanocyte. Recent studies have unequivocally demonstrated that Langerhans cells are independent cells with immune function. They display Fc and C3 receptors on their surface as well as la (immune region associated) antigens.

Human PMNs are phagocytosed by dendritic cells and interfere with DCs maturation: a new link between innate and acquired immunity

2010 ◽  
Vol 34 (8) ◽  
pp. S72-S72
Author(s):  
Qi Xu ◽  
Xiumin Ma ◽  
Yuejie Zhu ◽  
Fengsen Li ◽  
Jianbing Ding
Keyword(s):  
Dendritic Cells ◽  
Acquired Immunity ◽  
Human Pmns
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