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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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Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures using murine BM-DC and T-cell receptor transgenic OT-I/II T-cells.(A) Murine BM-DCs were matured for 4 hours, contacted with naïve syngeneic CD4+ enriched OT-II T-cells for 12 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with BM-DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 3 days. (B) Total cell count of murine enriched CD4+ OT-II T-cells after 3 days DC/T-cell co-culture under short-term (white squares) or long-term (black squares) conditions with 4 hours LPS/IFN-γ matured BM-DCs is shown. Pooled data from 12 experiments are shown as median±SEM. (C) A mixture of CD8+OT-I and CD4+OT-II T-cells was used (OT-I:OT-II = 1∶4) for short-term (white squares) or long-term (black squares) DC/T-cell co-culture, but analyzed on day 3 separately using specific staining for CD4+ or CD8+ molecules (middle and right graphs, respectively). Pooled data from 5 experiments is shown.
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pone-0054879-g003: Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures using murine BM-DC and T-cell receptor transgenic OT-I/II T-cells.(A) Murine BM-DCs were matured for 4 hours, contacted with naïve syngeneic CD4+ enriched OT-II T-cells for 12 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with BM-DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 3 days. (B) Total cell count of murine enriched CD4+ OT-II T-cells after 3 days DC/T-cell co-culture under short-term (white squares) or long-term (black squares) conditions with 4 hours LPS/IFN-γ matured BM-DCs is shown. Pooled data from 12 experiments are shown as median±SEM. (C) A mixture of CD8+OT-I and CD4+OT-II T-cells was used (OT-I:OT-II = 1∶4) for short-term (white squares) or long-term (black squares) DC/T-cell co-culture, but analyzed on day 3 separately using specific staining for CD4+ or CD8+ molecules (middle and right graphs, respectively). Pooled data from 5 experiments is shown.

Mentions: The same proliferation pattern as in the human test system was observed in murine DC/T-cell co-cultures (figure 3B). The cell number of OT-II CD4+ T-cells following short-term co-cultivation was significantly higher than following long-term co-cultivation (p = 0.0002; table 3). Next we aimed at assessing the effect of CD4+ helper T-cell priming on OT-I CD8+ cytolytic T-cells in a 1∶4 mixture in short-term co-culture with 6 hours matured DCs. We observed enhanced T-cell numbers in short-term compared to long-term co-cultures (OT-I CD8: p = 0.03; OT-II CD4: p = 0.0005; table 3; figure 3C). Similarly, when co-culturing 6 hours LPS/IFN-γ stimulated human DCs with total CD3+ human T-cells, proliferation of CD4+ as well as CD8+ T-cells was enhanced after short-term contact compared to continuous long-term contact conditions (CD4: p = 0.0003; CD8: p = 0.0001; table 3).


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 using murine BM-DC and T-cell receptor transgenic OT-I/II T-cells.(A) Murine BM-DCs were matured for 4 hours, contacted with naïve syngeneic CD4+ enriched OT-II T-cells for 12 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with BM-DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 3 days. (B) Total cell count of murine enriched CD4+ OT-II T-cells after 3 days DC/T-cell co-culture under short-term (white squares) or long-term (black squares) conditions with 4 hours LPS/IFN-γ matured BM-DCs is shown. Pooled data from 12 experiments are shown as median±SEM. (C) A mixture of CD8+OT-I and CD4+OT-II T-cells was used (OT-I:OT-II = 1∶4) for short-term (white squares) or long-term (black squares) DC/T-cell co-culture, but analyzed on day 3 separately using specific staining for CD4+ or CD8+ molecules (middle and right graphs, respectively). Pooled data from 5 experiments is shown.
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pone-0054879-g003: Proliferative capacity and polarization profile in short- versus long-term DC/T-cell co-cultures using murine BM-DC and T-cell receptor transgenic OT-I/II T-cells.(A) Murine BM-DCs were matured for 4 hours, contacted with naïve syngeneic CD4+ enriched OT-II T-cells for 12 hours, separated using flow sorting, and either maintained alone in culture or re-cultivated with BM-DCs (further-on referred to as “short”-term or “long”-term DC/T-cell interaction, respectively), both for 3 days. (B) Total cell count of murine enriched CD4+ OT-II T-cells after 3 days DC/T-cell co-culture under short-term (white squares) or long-term (black squares) conditions with 4 hours LPS/IFN-γ matured BM-DCs is shown. Pooled data from 12 experiments are shown as median±SEM. (C) A mixture of CD8+OT-I and CD4+OT-II T-cells was used (OT-I:OT-II = 1∶4) for short-term (white squares) or long-term (black squares) DC/T-cell co-culture, but analyzed on day 3 separately using specific staining for CD4+ or CD8+ molecules (middle and right graphs, respectively). Pooled data from 5 experiments is shown.
Mentions: The same proliferation pattern as in the human test system was observed in murine DC/T-cell co-cultures (figure 3B). The cell number of OT-II CD4+ T-cells following short-term co-cultivation was significantly higher than following long-term co-cultivation (p = 0.0002; table 3). Next we aimed at assessing the effect of CD4+ helper T-cell priming on OT-I CD8+ cytolytic T-cells in a 1∶4 mixture in short-term co-culture with 6 hours matured DCs. We observed enhanced T-cell numbers in short-term compared to long-term co-cultures (OT-I CD8: p = 0.03; OT-II CD4: p = 0.0005; table 3; figure 3C). Similarly, when co-culturing 6 hours LPS/IFN-γ stimulated human DCs with total CD3+ human T-cells, proliferation of CD4+ as well as CD8+ T-cells was enhanced after short-term contact compared to continuous long-term contact conditions (CD4: p = 0.0003; CD8: p = 0.0001; table 3).

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