scholarly journals Flow Cytometry-Based Analysis and Sorting of Lung Dendritic Cells

10.5772/37896 ◽  
2012 ◽  
Author(s):  
Svetlana P.
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells

scholarly journals FRI0015 PHENOTYPE AND FUNCTION OF THE PERIPHERAL BLOOD DENDRITIC CELLS OF PSORIASIS PATIENTS WITH AND WITHOUT ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 578.1-579
Author(s):  
S. Schnitte ◽  
A. Fuchs ◽  
T. Funk ◽  
A. C. Pecher ◽  
D. Dörfel ◽  
...  
Keyword(s):  
Risk Factors ◽  
Flow Cytometry ◽  
Dendritic Cells ◽  
Psoriatic Arthritis ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Surface Proteins ◽  
Research Support ◽  
Cross Sectional ◽  
Cell Counts

Background:Psoriasis is a frequent skin disease that can appear with an arthritic manifestation in approximately 30% of the cases [1]. The underlying excessive immune reaction caused by pro-inflammatory cytokines can be triggered by several risk factors [2]. Various subgroups of Dendritic cells (DCs) in the skin play a crucial role in the induction of the dermal inflammatory response [3].Objectives:As the role of peripheral blood DCs remains unknown and the cause of an arthritic manifestation is still not completely understood [4], this project aimed to detect differences in phenotype or function of peripheral blood DCs in psoriatic patients with or without arthritis.Methods:We analyzed peripheral blood cells of 60 psoriasis patients with and without arthritis. Different DC subpopulations were detected by flow cytometry. Monocyte-derived DCs were cultured with or without Lipopolysaccharides to gain immature (iDC) and mature (mDC) cells. The DC phenotype was determined by staining with CD80, CD83, CD86, CD206, CCR7, CD1a, HLA-DR, CD40, GPN-MB, DC209 and CD14. Their T-cell stimulatory capability was analyzed by co-incubation with Carboxyfluorescein succinimidyl ester stained lymphocytes and the quantification of CD4+ T-lymphocytes afterwards. To measure the migration capacity DCs were seated into transwell chambers with a semipermeable membrane and partly supplemented with Macrophage Inflammatory Protein 3 Beta (Mip3b). Migrated cells were detected by flow cytometry. Measured cell counts were normalized to cell counts without Mip3b stimulation.Results:Comparing the factor of increase of migrated mDC counts due to mip3b stimulation, we detected a significant lower rate in samples of patients with arthritis (PsA) compared to those of patients without (Ps). Assays of mDCs without mip3b stimulation showed a significant higher count of migrated cells in the samples of the arthritic group [Figure 1]. Cell counts with Mip3b stimulation did vary slightly in the groups. The DC subpopulations and the expression of analyzed cell surface proteins did not show significant differences. The amounts of stimulated T-Lymphocytes did not differ significantly.Figure 1.Migration essay showing mDCs following Mip3b (+miß3b) as multiples of mDCs without stimulation (-mip3b). The factor of increase is significantly lower in patients with arthritis (PsA) compared to patients without (Ps). Absolute counts of migrated mDCs without Mip3b are significantly higher in the arthritic group. Cell counts with stimulation do not differ significantly (data not shown). N=24, p<0.05Conclusion:CCL19 (Mip3b) is a potent ligand to the CCR7 receptor inducing migration of DCs towards the lymphatic node [5]. The CCR7 amounts on the DC surface did not differ significantly in the groups. The mDCs without CCL19 stimulation migrated in higher amounts in samples of arthritic patients. Cell counts of stimulated DCs showed only slight differences. These results could be generated by a different appearance of the DCs of arthritic patients that might facilitate migration. Further experiments focusing on this aspect should be performed. A possible effect of disruptive factors (age, sex, medication…) needs to be clarified.References:[1]Henes, J.C., et al.,High prevalence of psoriatic arthritis in dermatological patients with psoriasis: a cross-sectional study.Rheumatol Int, 2014.34(2): p. 227-34.[2]Lee, E.B., et al.,Psoriasis risk factors and triggers.Cutis, 2018.102(5s): p. 18-20.[3]Kim, T.G., S.H. Kim, and M.G. Lee,The Origin of Skin Dendritic Cell Network and Its Role in Psoriasis.Int J Mol Sci, 2017.19(1).[4]Veale, D.J. and U. Fearon,The pathogenesis of psoriatic arthritis.Lancet, 2018.391(10136): p. 2273-2284.[5]Ricart, B.G., et al.,Dendritic cells distinguish individual chemokine signals through CCR7 and CXCR4.J Immunol, 2011.186(1): p. 53-61.Acknowledgments:This project was financially supported by Novartis Pharma GmbH.Disclosure of Interests:Sarah Schnitte Grant/research support from: Reaserch grant by Novartis, Alexander Fuchs: None declared, Tanja Funk: None declared, Ann-Christin Pecher: None declared, Daniela Dörfel: None declared, Jörg Henes Grant/research support from: Novartis, Roche-Chugai, Consultant of: Novartis, Roche, Celgene, Pfizer, Abbvie, Sanofi, Boehringer-Ingelheim,

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

Abstract MP27: Human Hypertension And Endothelial Cell Activation Promote The Formation And Activation Of Axl + Siglec-6 + Dendritic Cells Via Endothelial Release Of Growth Arrest Specific 6

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Justin P Van Beusecum ◽  
Natalia R Barbaro ◽  
Charles D Smart ◽  
David M Patrick ◽  
Cyndya A Shibao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Growth Arrest ◽  
Positive Association ◽  
Endothelial Cell Activation ◽  
Human Hypertension

We have shown that dendritic cells (DCs) from hypertensive mice convey hypertension when adoptively transferred to recipients. Recently a novel subset of DCs in humans that express Axl and Sigelc-6 + (AS DCs) have been identified which drive T cell proliferation and produce IL-1β, IL-6 and IL-23, consistent with DCs we have observed in hypertension. We hypothesized that AS cells are increased in hypertension and contribute to immune activation in this disease. We quantified circulating AS DCs by flow cytometry in normotensive (n=23) and hypertensive (n=11) subjects and found a more than 2-fold increase in circulating AS DCs in hypertensive compared to normotensive subjects (297 ± 73 vs. 108 ± 26/ml; p =0.0304). To investigate the mechanism by which AS DCs are formed in hypertension, we co-cultured human aortic endothelial cells (HAECs) undergoing either normotensive (5%) or hypertensive (10%) cyclical stretch for 48 hours with CD14 + monocytes from normotensive donors. Co-culture of monocytes with HAECs exposed to 10% stretch significantly increased AS DCs and AS DC IL-1β production when compared to 5% stretch alone as assessed by flow cytometry (21 ± 5 vs. 131 ± 32 IL-1β + AS DCs). Moreover, inhibition of Axl signaling with R248, completely abolished the production of IL-1β in AS DCs (34 ± 8 IL-1β + AS DCs). In additional experiments we found that 10% stretch caused a 50% increase in release of growth arrest 6 (GAS6), the ligand for Axl, from HAECs compared to 5% stretch. Treatment of human monocytes with GAS6 mimicked the effect of 10% stretch in promoting AS cell formation and IL-1β production. Based on the increased secretion of GAS6 from HAECs, we used a J-wire to harvest human endothelial cells from 23 additional volunteers to assess endothelial cell activation and GAS6 secretion in vivo. We found a positive association between pulse pressure and plasma GAS6 (R 2 =0.25, p =0.0079) and a striking positive association between GAS6 and ICAM-1 (R 2 =0.39, p =0.0012). These data show that secretion of GAS6 by an activated endothelial seems to promote the formation and activation of AS DCs. Thus, the interplay between endothelial-derived GAS6 and AS DCs seem to be an important mechanism in human hypertension and might be a novel therapeutic target for this disease.

Purification of human lung dendritic cells by four-color flow cytometry

1996 ◽  
pp. 1447-1454
Author(s):  
J AUBRY ◽  
S RISON ◽  
C POWER ◽  
J BONNEFOY
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Human Lung ◽  
Color Flow

Sub-populations of human spleen dendritic cells characterized by flow cytometry and in situ immunofluorescence

1997 ◽  
Vol 56 ◽  
pp. 266
Author(s):  
D. McIlroy ◽  
C. Troadec ◽  
J. Feuillard ◽  
B. Barrou ◽  
B. Autran ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Human Spleen

scholarly journals Understanding the Underlying Immune Response in HIT: Uptake of PF4/Heparin Complexes By Monocytes & Dendritic Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4197-4197
Author(s):  
Manali V. Joglekar ◽  
Sanjay Khandelwal ◽  
Mortimer Poncz ◽  
Lubica Rauova ◽  
Gowthami M Arepally
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Dendritic Cells ◽  
Cellular Uptake ◽  
Dependent Manner ◽  
Cellular Interactions ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Late Endosomes ◽  
Positively Charged

Abstract Platelet Factor 4 (PF4), a strongly positive-charged small protein, and the negatively-charged polymer heparin (Hep) form ultra-large complexes (ULCs) that have several unusual features including their remarkable stability and their ability to elicit a robust antibody response in vivo (Rauova L; Blood 2005 and Suvarna S, Blood 2005). Recently, we and others have shown that similar complexes of another positively charged protein, protamine sulfate with heparin can also lead to a clinically relevant immune response. To better understand the cellular basis for PF4/Hep antibody formation, we investigated mechanisms of cellular interactions and uptake of PF4 and PF4/Hep ULCs. For these studies, we examined cellular uptake of unlabeled or labeled PF4, heparin, or PF4/Hep using monocytes (PBMCs), dendritic cells (DCs) and/or neutrophils derived from peripheral blood. For these studies, cells were incubated with varying concentrations of unlabeled or fluorescently-labeled antigen (PF4, Hep or PF4/Hep ULCs). In cellular studies using unlabeled antigen, uptake was detected by fluorescently-labeled KKO, a monoclonal antibody to PF4/Hep complexes. Cellular uptake was visualized by confocal microscopy or flow cytometry. In initial studies, we defined the time course of uptake. As shown in Figure 1, we demonstrate that PF4/Hep-FITC ULCs are taken up by PBMCs in a time-dependent manner, with maximal uptake occurring between 12-24 hours (Figure 1, only 24 hour time point shown). This uptake is independent of the fluorescent label, as labeled or unlabeled intracellular PF4/Hep ULCs were readily visualized by KKO-AF647. To examine the effect of Hep on PF4 uptake, PBMCs were incubated with unlabeled PF4 alone or in the presence of increasing concentrations of Hep-FITC (0.1-2.5 U/mL). As shown in Figure 2A, Hep markedly enhances the efficiency of cellular uptake of PF4 in a Hep-dependent manner. Increased number of intracellular vesicles containing labeled PF4/Hep-FITC was noted at Hep-FITC concentration of 0.25-1 U/mL (Figure 2B; fluorescent vesicles/cell: 0.6 ± 0.22 for 0.35 U/mL and 0.5 ± 0.26 for 1U/mL) as compared to PF4 alone (25 µg/mL; number of fluorescent vesicles/cell: 0.35 ± 0.07). On examining the uptake of ULCs by other phagocytic cells, we could not demonstrate PF4/Hep uptake by neutrophils, suggesting that only monocytes/DCs provide clearance of complexes. Cellular uptake of Hep-containing ULCs was not limited to complexes of PF4 and heparin but also other positively-charged proteins, as intracellular complexes could be demonstrated when Hep-FITC was incubated with murine PF4, protamine, or lysozyme to form corresponding protein/Hep-FITC ULCs. This uptake was an active process of monocytes as PF4/Hep ULC endocytosis was inhibited by 4C, and cytochalasin D, an actin polymerization inhibitor and was associated with cellular activation and expression of MHC II and CD83 co-stimulatory molecules as shown by flow cytometry (Figure 3). Finally, co-staining with KKO and lysosomal associated membrane protein-2 (LAMP-2) localized intact PF4/Hep ULCs into late endosomes. Taken together, these studies demonstrate that PF4/Hep and other protein/heparin ULCs are taken up actively by monocytes and/or DCs, intact ULCs can be detected in late endosomes and uptake is accompanied by cellular activation. These studies establish a distinct role for heparin in increasing the uptake and cellular activation of PF4 and other positively charged complexes. These studies additionally provide insights into why the majority of clinical cases of HIT occur in the wake of heparin exposure. Disclosures Arepally: TEVA Pharma: Consultancy.

scholarly journals Comprehensive Phenotyping of Dendritic Cells in Cancer Patients by Flow Cytometry

2020 ◽  
Author(s):  
Claudia Carenza ◽  
Sara Franzese ◽  
Francesca Calcaterra ◽  
Domenico Mavilio ◽  
Silvia Della Bella
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Cancer Patients

Inhibition of mTOR suppresses IFNα production and the STING pathway in monocytes from systemic lupus erythematosus patients

Rheumatology ◽  
2020 ◽  
Vol 59 (10) ◽  
pp. 2992-3002 ◽  
Author(s):  
Goh Murayama ◽  
Asako Chiba ◽  
Taiga Kuga ◽  
Ayako Makiyama ◽  
Ken Yamaji ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Intracellular Cytokine Staining ◽  
Plasmacytoid Dendritic Cells ◽  
Mtor Pathway ◽  
Pathway Activation ◽  
Sting Pathway

Abstract Objective Increased IFNα is important in the pathogenesis of SLE. Plasmacytoid dendritic cells are considered the main producer of IFNα upon Toll-like receptor pathway activation. However, which cells produce IFNα following stimulation with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) in SLE remains unknown. We investigated the IFNα producing capacity of myeloid cells under cGAS-STING pathway stimulation. Methods IFNα levels in peripheral blood mononuclear cells from SLE patients and healthy controls stimulated with 2′3′c-GAMP, a stimulator of cGAS-STING, were measured by intracellular cytokine staining and flow cytometry. STING expression and its co-localization with TBK1 were examined by flow cytometry or confocal microscopy. The effects of in vitro exposure to IFNα on IFNα production and STING expression, and in vitro rapamycin treatment on IFNα production and STING, pTBK1 and IRF3 expression were examined. Results IFNα was produced by monocytes, conventional dendritic cells and plasmacytoid dendritic cells upon cGAS-STING pathway activation. The frequency of IFNα-producing monocytes positively correlated with SLE disease activity. STING expression and its co-localization with TBK1 were increased in lupus monocytes. Prior exposure to IFNα enhanced the IFNα-producing capacity of monocytes. Inhibition of the mechanistic target of the rapamycin (mTOR) pathway suppressed IFNα production from monocytes and downregulated enhanced STING expression and its downstream molecules. Conclusion Enhanced IFNα from lupus monocytes induced by augmented STING pathway activation is associated with SLE pathogenesis. Suppression of the mTOR pathway downregulated the enhanced STING expression and the subsequent IFNα production by monocytes.

Decreased Expression of Indoleamine 2,3-Dioxygenase 1 (IDO 1) in Dendritic Cells From Patients with Immune Thrombocytopenia (ITP) Correlates with Impaired Regulatory T Cells Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2770-2770
Author(s):  
Lucia Catani ◽  
Daria Sollazzo ◽  
Antonio Curti ◽  
Sara Trabanelli ◽  
Cecilia Evangelisti ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
Regulatory T Cells ◽  
Healthy Subjects ◽  
Effector T Cells ◽  
Pathogenetic Role ◽  
Chronic Itp ◽  
Ido1 Expression

Abstract Abstract 2770 Recent studies suggest a bi-directional interaction between regulatory T cells (Tregs) and dendritic cells (DCs). Despite their role as inducers of immunity, DCs may also be critical in maintaining tolerance to self-antigens by inducing Tregs. Specifically, Indoleamine 2,3-dioxygenase 1 (IDO 1) expressing DCs expand Tregs. In turn, Tregs, which express CTLA4, engage its receptors B7-1(CD80)/B7-2(CD86), thus inducing the expression of IDO1 enzyme in DCs. IDO1 expression leads to tryptophan depletion and immunosuppressive kynurenine enrichment in the tissue. Both factors drive the de novo generation of Tregs from CD4+ T cells. In addition, Tregs may modulate the maturation and/or function of DCs, which are required for the activation of effector T cells. Tregs out compete with naïve T cells in aggregating around DCs. After forming aggregates, Tregs specifically downregulate the expression of CD80 and CD86 on DCs. The result of the interaction between Tregs and DCs is the impaired capacity of the antigen presenting cells to establish long lasting interactions with effector T cells. Therefore, given the crucial role of DCs and Tregs in initiating, regulating, maintaining or repressing immune responses, investigations on their interaction is of great importance for better elucidating the pathogenesis of ITP. Of note, in ITP this interaction and its pathogenetic role have never been investigated in depth. In this paper, we enumerated and functionally characterized Treg subsets, exploring whether in ITP abnormal Tregs interactions with DCs and/or effector T cells might play a pathogenetic role. Specifically, we studied whether, in ITP patients: 1) DCs maturation is differentially modulated by Tregs from ITP patients as compared with healthy donors; 2) the mechanism of Tregs generation is altered. Forty adult ITP patients, newly diagnosed (14 cases) or with persistent (20 cases) or chronic ITP (9 cases) were studied after informed consent. At the time of the study, patients with persistent or chronic ITP were out off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 53×109/L (range 8–99). Allogeneic Mixed Leukocyte Reaction (MLR) was performed to test the suppressive activity of Tregs. To analyze the in vitro ability of highly purified CD4+CD25+ T cells to inhibit DCs maturation, normal immature CD14-derived DCs were cultured alone and with allogeneic CD4+CD25+ T cells from healthy subjects or ITP patients in the presence of Lipopolysaccaryde. CD80 and CD86 expression of CD14-derived DCs was then tested at flow cytometry. To evaluate the in vitro conversion of non Tregs into Tregs, CD4+CD25- T cells were cultured alone and with autologous mature CD14-derived DCs from ITP patients and healthy subjects. The percentages of CD4+CD25highFoxP3+ T cells were quantitated at flow cytometry. mRNA IDO1 expression of immature and mature CD14-derived DCs was then evaluated by Real-time RT-PCR. To determine IDO1 enzyme activity kynurenine levels were measured in the supernantants of mature CD14-derived DCs. We found that in ITP Tregs show lower ability to suppress T cell proliferation and to inhibit CD14-derived DCs maturation because they do not affect the expression of the costimulatory molecules CD80 and CD86. We show that the absolute number of Tregs was significantly decreased in ITP patients in comparison to healthy subjects (CD4+CD25highFoxp3+ T cells (5.5±4.3 vs 11.6±6.9 cells/microL; p below 0.01) and CD4+CD25highCD127low/negative T cells (50.9±27.3 vs 80.5±37.7 cells/microL; p below 0.02)). In addition, in ITP patients we document that the low number of circulating Tregs may be due to the reduced ability of mature CD14-derived DCs to convert non-Treg cells (CD4+CD25-) into Tregs (CD4+CD25highFoxP3+ Tregs). This is related to reduced expression and activity of the IDO1 enzyme in mature CD14-derived DCs from ITP patients, since DCs expressing IDO1 favour Treg generation. In conclusion, taken together our data demonstrate that in ITP the cross-talk between Tregs and DCs is impaired and plays a pathogenetic role. As a consequence, we found the generation of more immunogenic DCs and defective Tregs. Disclosures: No relevant conflicts of interest to declare.

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