Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood

Bender, Armin; Sapp, Mark; Schuler, Gerold; Steinman, Ralph M.; Bhardwaj, Nina

doi:10.1016/0022-1759(96)00079-8

Cited by 622 publications

(430 citation statements)

References 32 publications

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“…4,28 Owing to these features and establishment of methods for expansion of human and rodent DCs on a large scale from hematopoietic precursors in the presence of appropriate cytokine cocktails, [29][30][31] DCs have attracted great attention as vehicles for the delivery of cancer vaccines. The efficacy of different viral vectors, including retroviral vector, 8,32 Ad, [8][9][10][11][12] and lentiviral vector, 33,34 has been evaluated for genetic modification of DCs.…”

Section: Discussionmentioning

confidence: 99%

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

et al. 2003

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Dendritic cells (DCs) are the most potent professional antigen-presenting cells for the initiation of antigen-specific immune responses, and antigen-loaded DCs have been regarded as promising vaccines in cancer immunotherapy. We previously demonstrated that RGD fiber-mutant adenovirus vector (AdRGD) could attain highly efficient gene transduction into human and murine DCs. The aim of the present study is to demonstrate the predominance of ex vivo genetic DC manipulation using AdRGD in improving the efficacy of DC-based immunotherapy targeting gp100, a melanoma-associated antigen (MAA). Vaccination with murine bone marrow-derived DCs transduced with AdRGD encoding gp100 (AdRGD-gp100/mBM-DCs) dramatically improved resistance to B16BL6 melanoma challenge and pulmonary metastasis as compared with immunization with conventional Ad-gp100-transduced mBM-DCs. The improvement in antimelanoma effects upon immunization with AdRGD-gp100/mBM-DCs correlated with enhanced cytotoxic activities of natural killer (NK) cells and B16BL6-specific cytotoxic T lymphocytes (CTLs). Furthermore, in vivo depletion analysis demonstrated that CD8 þ CTLs and NK cells were the predominant effector cells responsible for the anti-B16BL6 immunity induced by vaccination with AdRGDgp100/mBM-DCs, and that helper function of CD4 þ T cells was necessary for sufficiently eliciting effector activity. These findings clearly revealed that highly efficient MAA gene transduction to DCs by AdRGD could greatly improve the efficacy of DC-based immunotherapy against melanoma.

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Section: Discussionmentioning

confidence: 99%

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

et al. 2003

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“…To generate BMDC total BM cells were cultured in medium containing murine GM-CSF (1000 U/mL) for 7-9 days, as previously described [44]. DC yield was monitored by staining with anti-CD11b, anti-CD11c antibodies (BD/Pharmingen) and anti-Dectin-1 (Serotec).…”

Section: Culturesmentioning

confidence: 99%

Requirement of phospholipase C‐γ2 (PLCγ2) for Dectin‐1‐induced antigen presentation and induction of T_H1/T_H17 polarization

et al. 2009

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DC recognize microbial components through an array of receptors known as PRR. PRR initiate intracellular signals, which engender DC with the capacity to stimulate T-cell responses. Dectin-1 is a PRR that recognizes b-glucan, a major constituent of many fungi's outer cell wall. Here we show that Dectin-1 activates DC through phospholipase (PLC)c2 signaling. PLCc2-deficient DC were unable to expand antigen-specific T cells and induce T H 1 and T H 17 differentiation in response to b-glucan. Mechanistically, PLCc2-deficiency impaired the capacity of DC to secrete polarizing cytokines following exposure to b-glucan. Dectin-1 required PLCc2 to activate MAPK, AP-1 and NF-jB, which induce cytokine gene expression. Moreover, PLCc2 controlled Dectin-1-mediated NFAT activation and induction of NFAT-dependent genes such as IL-2, cyclooxigenase-2 and Egr transcription factors. We conclude that PLCc2 is a crucial signaling mediator that modifies DC gene expression program to activate DC responses to b-glucan-containing pathogens.Key words: DC . Dectin-1 . PLCg . Signaling Supporting Information available online Introduction DC play a key role in gathering information from the surrounding environment and initiating appropriate immune responses when needed [1,2]. Because they bridge innate and adaptive immunity, DC are equipped with a multitude of intracellular and cell surface receptors that sense microbial components and trigger host responses to invading pathogens. These receptors, collectively known as PRR, include TLR [3], C-type lectins such as Dectin-1 [4], scavenger receptors [5], the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2, the NOD-like receptors and the RIG-like receptors [6].Dectin-1 recognizes b-glucan, a major constituent of many fungi's outer cell wall [4,7] and triggers intracellular signals through a cytoplasmic tyrosine motif resembling the ITAM. Upon ligand binding, this motif is phosphorylated by src family kinases [8] and recruits the tyrosine kinase Syk [9], which initiates a signaling cascade leading to NF-kB [10], NFAT [11] and MAPK [12] activation. Along with Bcl-10 and MALT1, CARD9 is a crucial mediator in the ITAM-signaling pathway that links recognition of b-glucan by Dectin-1 to NF-kB and MAPK activation [13,14]. Overall, the Syk-CARD9 pathway is essential for [20,21]. Syk, together with the tyrosine kinases BTK and Src, activate PLCg through tyrosine phosphorylation [22]. Another downstream ITAM mediator, PI-3K, activates PLCg by generating phosphatidylinositol 3,4,5 trisphosphate, which binds the pleckstrin homology domain of PLCg, promoting PLCg recruitment to the cell membrane. Thus, the ability of Dectin-1 to initiate an ITAM-Syk-signaling pathway [9] provides a rationale for studying the role of PLCg in Dectin-1-mediated antimicrobial responses in DC.PLCg includes two isoforms, PLCg1 and PLCg2, which hydrolyze membrane phosphatidylinositol 4,5-biphosphate into inositol 1,4,5-trisphosphate and diacylglicerol (DAG) [20]. Inositol 1,4,5-trisphosphate triggers the ...

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“…Monocyte-derived DCs were generated from the adherent fraction of PBMCs obtained from healthy control subjects, as described (Bender et al, 1996). Briefly, PBMCs were allowed to adhere for 2 h in culture flasks, nonadherent cells were gently removed, and the remaining cells were cultured in RPMI 1640 containing 10% heatinactivated FCS (Euroclone, Wetherby, West York, UK) and supplemented with 800 U ml À1 GM-CSF (Novartis, Italy) and 10 ng ml À1 IL-4 (PeproTech, London, UK).…”

Section: Effects Of Spermine On In Vitro Cultured Dcsmentioning

confidence: 99%

Altered maturation of peripheral blood dendritic cells in patients with breast cancer

et al. 2003

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Tumours have at least two mechanisms that can alter dendritic cell (DC) maturation and function. The first affects the ability of haematopoietic progenitors to differentiate into functional DCs; the second affects their differentiation from CD14 þ monocytes, promoting an early but dysfunctional maturation. The aim of this study was to evaluate the in vivo relevance of these pathways in breast cancer patients. For this purpose, 53 patients with invasive breast cancer were compared to 68 healthy controls. To avoid isolation or culture procedures for enrichment of DCs, analyses were directly performed by flow cytometry on whole-blood samples. The expression of surface antigens and intracellular accumulation of regulatory cytokines upon LPS stimulation were evaluated. The number of DCs, and in particular of the myeloid subpopulation, was markedly reduced in cancer patients (Po0.001). Patient DCs were characterized by a more mature phenotype compared with controls (P ¼ 0.016), and had impaired production of IL-12 (Po0.001). These alterations were reverted by surgical resection of the tumour. To investigate the possible role of some tumourrelated immunoactive soluble factors, we measured the plasmatic levels of vascular endothelial growth factor, IL-10 and spermine. A significant inverse correlation between spermine concentration and the percentage of DCs expressing IL-12 was found. Evidence was also obtained that in vitro exposure of monocyte-derived DCs to spermine promoted their activation and maturation, and impaired their function. Taken together, our results suggest that both the above-described mechanisms could concomitantly act in breast cancer to affect DC differentiation, and that spermine could be a mediator of dysfunctional maturation of DCs.

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Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood

Cited by 622 publications

References 32 publications

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

Requirement of phospholipase C‐γ2 (PLCγ2) for Dectin‐1‐induced antigen presentation and induction of T_H1/T_H17 polarization

Altered maturation of peripheral blood dendritic cells in patients with breast cancer

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Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood

Cited by 622 publications

References 32 publications

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

Requirement of phospholipase C‐γ2 (PLCγ2) for Dectin‐1‐induced antigen presentation and induction of TH1/TH17 polarization

Altered maturation of peripheral blood dendritic cells in patients with breast cancer

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Requirement of phospholipase C‐γ2 (PLCγ2) for Dectin‐1‐induced antigen presentation and induction of T_H1/T_H17 polarization