Induced cytokine response of human PMBC-cultures: Correlation of gene expression and secretion profiling and the effect of cryopreservation

Radke, Lars; Hemmerling, Diana A. López; Lubitz, Annika; Giese, Christoph; Frohme, Marcus

doi:10.1016/j.cellimm.2011.10.018

Cited by 11 publications

(6 citation statements)

References 34 publications

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“…The cytokine profiles induced by stimulation of lymph node cells from naïve ferrets with mitogens were consistent with those reported in studies using human and other animal leukocytes. Stimulation of ferret lymph node cells with ConA induced similar profiles to cultures of mouse splenocytes (Candolfi et al, 1995), human PBMCs (Al Wabel et al, 1993;Yaqoob and Calder, 1998;Radke et al, 2012), woodchuck PMBCs (Menne et al, 2002) and calf CD4 + and CD8 + PBMCs (Tanaka et al, 2007) stimulated with ConA. Of interest, ConA has been shown to induce expression of IL1␤ and low levels of IL1␣ in human PBMCs (Yaqoob and Calder, 1998), but we did not detect increases in either cytokine in ferret lymph node cells.…”

Section: Discussionmentioning

confidence: 60%

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

Carolan

Butler

Rockman

et al. 2014

Journal of Virological Methods

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The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.

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Section: Discussionmentioning

confidence: 60%

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

Carolan

Butler

Rockman

et al. 2014

Journal of Virological Methods

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“…IL-2 levels induced by stimulation with xylosylated RSV-F are continuously present in the first two weeks. However, IL-2 levels are relatively low for both vaccines, compared to studies with other stimulants [ 32 ]. Finally, GM-CSF peaks are higher (e.g., 6.9 fold and 9.0 fold higher at day 22 and 23) in the days after restimulation for cells treated with RSV-F Xyl+, indicating an enhancing effect on the cellular response and adaptive immunity, since monocytes can mature into dendritic cells.…”

Section: Resultsmentioning

confidence: 99%

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans

et al. 2017

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Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

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“…In the cell culture stimulation experiment CD14 (−) PBMC were co-cultured with mDC. Therefore, CD14 (+) and CD14 (−) cells were enriched from whole blood of healthy donors, as described before [ 21 ]. Immature dendritic cells (iDC) were differentiated from enriched CD14 (+) cells by the addition of interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), 800 U/mL each (both Miltenyi Biotec, Bergisch Gladbach, Germany), on day 1 and 2.…”

Section: Methodsmentioning

confidence: 99%

“…The concentrations of six key cytokines were quantified with a bead-based suspension assay (BioPlex, BioRad). These key cytokines were identified in previous studies [ 21 ] and cover cellular (IL-2) and humoral (IL-4) immune response, as well as pro- (IFN-g, TNF-a) and anti-inflammatory (IL-10) cytokines and growth factors (GM-CSF). Since cells can only be harvested at the end of the trial, the same experiment was conducted in a fed batch microtiter plate with a comparable stimulation pattern.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor

Radke

Sandig

Lubitz³

et al. 2017

Bioengineering

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Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

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Induced cytokine response of human PMBC-cultures: Correlation of gene expression and secretion profiling and the effect of cryopreservation

Cited by 11 publications

References 34 publications

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor

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