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Toll-like receptor 4 engagement drives differentiation of human and murine dendritic cells from a pro- into an anti-inflammatory mode.

Luger R, Valookaran S, Knapp N, Vizzardelli C, Dohnal AM, Felzmann T - PLoS ONE (2013)

Bottom Line: T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels.When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity.We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response.

View Article: PubMed Central - PubMed

Affiliation: St. Anna Children's Cancer Research Institute, Laboratory of Tumor Immunology, Department of Pediatrics, Medical University Vienna, Austria.

ABSTRACT
The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs' initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

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Related in: MedlinePlus

Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures.(A) Human DCs were matured for 6 or 48 hours, contacted with naïve allogeneic CD4+ enriched T-cells for 6 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 6 days. After the primary short- or long-term DC/T-cell co-cultures, T-cells were analyzed for FoxP3 expression or, adjusted to equal cell numbers, were re-stimulated with anti-CD3 for 24 hours. The supernatant of the re-stimulated culture was further analyzed for polarization-specific cytokine release. DCs matured for 6 (B) or 48 hours (C) with LPS, LPS/IFN-γ, or mediator cocktail (TNF-α/PG-E2/IL-1β/IL-6) were put in short-term (white squares) or long-term (black squares) co-culture with CD4+ T-cells. (i) Absolute numbers of proliferating CD4+CFSE−T-cells after 6 days of DC/T-cell co-culture are given. Pooled data from 8 to 10 experiments are shown. (ii) The cytokine composition in the supernatant of the 6 hours T-cell priming co-cultures with 6 or 48 hours matured DCs before sorting are shown. Asterisks indicate cytokine levels below the detection limit of the assay. Pooled data from 3 to 5 experiments are shown as median±SEM. Asterisks indicate cytokine levels below the detection limit of the assay. (iii) The culture supernatants of anti-CD3 re-stimulated CD4+ T-cells from short- (white squares) or long-term (black squares) primary DC/T-cell co-cultures with LPS/IFN-γ matured DCs (either 6 or 48 hours matured) were analyzed for their content of IFN-γ, IL-4, IL-17A, and IL-10. Three to 10 independent experiments are shown. (iv) Detection of FoxP3 in T-cells after short- or long-term DC/T-cell co-culture with LPS/IFN-γ stimulated DCs (matured for 6 or 48 hours). As a positive control (+) the cell lysate of a FoxP3 transfected cell line was used. One representative out of 3 experiments is shown. All data is given as median±SEM. DCs from Donors C, E and F were used in experiments (A) to (C).
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pone-0054879-g002: Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures.(A) Human DCs were matured for 6 or 48 hours, contacted with naïve allogeneic CD4+ enriched T-cells for 6 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 6 days. After the primary short- or long-term DC/T-cell co-cultures, T-cells were analyzed for FoxP3 expression or, adjusted to equal cell numbers, were re-stimulated with anti-CD3 for 24 hours. The supernatant of the re-stimulated culture was further analyzed for polarization-specific cytokine release. DCs matured for 6 (B) or 48 hours (C) with LPS, LPS/IFN-γ, or mediator cocktail (TNF-α/PG-E2/IL-1β/IL-6) were put in short-term (white squares) or long-term (black squares) co-culture with CD4+ T-cells. (i) Absolute numbers of proliferating CD4+CFSE−T-cells after 6 days of DC/T-cell co-culture are given. Pooled data from 8 to 10 experiments are shown. (ii) The cytokine composition in the supernatant of the 6 hours T-cell priming co-cultures with 6 or 48 hours matured DCs before sorting are shown. Asterisks indicate cytokine levels below the detection limit of the assay. Pooled data from 3 to 5 experiments are shown as median±SEM. Asterisks indicate cytokine levels below the detection limit of the assay. (iii) The culture supernatants of anti-CD3 re-stimulated CD4+ T-cells from short- (white squares) or long-term (black squares) primary DC/T-cell co-cultures with LPS/IFN-γ matured DCs (either 6 or 48 hours matured) were analyzed for their content of IFN-γ, IL-4, IL-17A, and IL-10. Three to 10 independent experiments are shown. (iv) Detection of FoxP3 in T-cells after short- or long-term DC/T-cell co-culture with LPS/IFN-γ stimulated DCs (matured for 6 or 48 hours). As a positive control (+) the cell lysate of a FoxP3 transfected cell line was used. One representative out of 3 experiments is shown. All data is given as median±SEM. DCs from Donors C, E and F were used in experiments (A) to (C).

Mentions: The sequential secretion of IL-12 and IL-10 suggested that limiting the priming of T-cells by DCs to the time when IL-12 is predominantly secreted and before IL-10 starts accumulating, favors Th1 polarization and CTL priming. A brief exposure to LPS with and without IFN-γ was used to trigger maturation in human (6 hours) and murine (4 hours) DCs, after which DC/T-cell co-cultures were initiated. All human DC/T-cell co-cultures were discontinued after 6 hours in order to separate T-cells from DCs using flow sorting. Murine T-cells were more sensitive to purification and flow sorting procedures in preliminary experiments, which lead us to prolong murine DC/T-cell co-cultures up to 12 hours before flow sorting T-cells from DCs. This extended contact did not change the net-outcome of the experiments (data not shown). The purified T-cells were then either re-cultivated alone (here termed “short-term co-culture”) or together with the sorted DCs (here termed “long-term co-culture”, figure 2); 6 days in the human system or 3 days in the murine system.


Toll-like receptor 4 engagement drives differentiation of human and murine dendritic cells from a pro- into an anti-inflammatory mode.

Luger R, Valookaran S, Knapp N, Vizzardelli C, Dohnal AM, Felzmann T - PLoS ONE (2013)

Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures.(A) Human DCs were matured for 6 or 48 hours, contacted with naïve allogeneic CD4+ enriched T-cells for 6 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 6 days. After the primary short- or long-term DC/T-cell co-cultures, T-cells were analyzed for FoxP3 expression or, adjusted to equal cell numbers, were re-stimulated with anti-CD3 for 24 hours. The supernatant of the re-stimulated culture was further analyzed for polarization-specific cytokine release. DCs matured for 6 (B) or 48 hours (C) with LPS, LPS/IFN-γ, or mediator cocktail (TNF-α/PG-E2/IL-1β/IL-6) were put in short-term (white squares) or long-term (black squares) co-culture with CD4+ T-cells. (i) Absolute numbers of proliferating CD4+CFSE−T-cells after 6 days of DC/T-cell co-culture are given. Pooled data from 8 to 10 experiments are shown. (ii) The cytokine composition in the supernatant of the 6 hours T-cell priming co-cultures with 6 or 48 hours matured DCs before sorting are shown. Asterisks indicate cytokine levels below the detection limit of the assay. Pooled data from 3 to 5 experiments are shown as median±SEM. Asterisks indicate cytokine levels below the detection limit of the assay. (iii) The culture supernatants of anti-CD3 re-stimulated CD4+ T-cells from short- (white squares) or long-term (black squares) primary DC/T-cell co-cultures with LPS/IFN-γ matured DCs (either 6 or 48 hours matured) were analyzed for their content of IFN-γ, IL-4, IL-17A, and IL-10. Three to 10 independent experiments are shown. (iv) Detection of FoxP3 in T-cells after short- or long-term DC/T-cell co-culture with LPS/IFN-γ stimulated DCs (matured for 6 or 48 hours). As a positive control (+) the cell lysate of a FoxP3 transfected cell line was used. One representative out of 3 experiments is shown. All data is given as median±SEM. DCs from Donors C, E and F were used in experiments (A) to (C).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569454&req=5

pone-0054879-g002: Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures.(A) Human DCs were matured for 6 or 48 hours, contacted with naïve allogeneic CD4+ enriched T-cells for 6 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 6 days. After the primary short- or long-term DC/T-cell co-cultures, T-cells were analyzed for FoxP3 expression or, adjusted to equal cell numbers, were re-stimulated with anti-CD3 for 24 hours. The supernatant of the re-stimulated culture was further analyzed for polarization-specific cytokine release. DCs matured for 6 (B) or 48 hours (C) with LPS, LPS/IFN-γ, or mediator cocktail (TNF-α/PG-E2/IL-1β/IL-6) were put in short-term (white squares) or long-term (black squares) co-culture with CD4+ T-cells. (i) Absolute numbers of proliferating CD4+CFSE−T-cells after 6 days of DC/T-cell co-culture are given. Pooled data from 8 to 10 experiments are shown. (ii) The cytokine composition in the supernatant of the 6 hours T-cell priming co-cultures with 6 or 48 hours matured DCs before sorting are shown. Asterisks indicate cytokine levels below the detection limit of the assay. Pooled data from 3 to 5 experiments are shown as median±SEM. Asterisks indicate cytokine levels below the detection limit of the assay. (iii) The culture supernatants of anti-CD3 re-stimulated CD4+ T-cells from short- (white squares) or long-term (black squares) primary DC/T-cell co-cultures with LPS/IFN-γ matured DCs (either 6 or 48 hours matured) were analyzed for their content of IFN-γ, IL-4, IL-17A, and IL-10. Three to 10 independent experiments are shown. (iv) Detection of FoxP3 in T-cells after short- or long-term DC/T-cell co-culture with LPS/IFN-γ stimulated DCs (matured for 6 or 48 hours). As a positive control (+) the cell lysate of a FoxP3 transfected cell line was used. One representative out of 3 experiments is shown. All data is given as median±SEM. DCs from Donors C, E and F were used in experiments (A) to (C).
Mentions: The sequential secretion of IL-12 and IL-10 suggested that limiting the priming of T-cells by DCs to the time when IL-12 is predominantly secreted and before IL-10 starts accumulating, favors Th1 polarization and CTL priming. A brief exposure to LPS with and without IFN-γ was used to trigger maturation in human (6 hours) and murine (4 hours) DCs, after which DC/T-cell co-cultures were initiated. All human DC/T-cell co-cultures were discontinued after 6 hours in order to separate T-cells from DCs using flow sorting. Murine T-cells were more sensitive to purification and flow sorting procedures in preliminary experiments, which lead us to prolong murine DC/T-cell co-cultures up to 12 hours before flow sorting T-cells from DCs. This extended contact did not change the net-outcome of the experiments (data not shown). The purified T-cells were then either re-cultivated alone (here termed “short-term co-culture”) or together with the sorted DCs (here termed “long-term co-culture”, figure 2); 6 days in the human system or 3 days in the murine system.

Bottom Line: T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels.When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity.We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response.

View Article: PubMed Central - PubMed

Affiliation: St. Anna Children's Cancer Research Institute, Laboratory of Tumor Immunology, Department of Pediatrics, Medical University Vienna, Austria.

ABSTRACT
The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs' initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.

Show MeSH
Related in: MedlinePlus