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Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells.

Veldhoen M, Hirota K, Christensen J, O'Garra A, Stockinger B - J. Exp. Med. (2008)

Bottom Line: Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR).However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells.The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR). In this study, we show that differentiation of Th17 cells, but not Th1 or induced regulatory T (iT reg) cells, is increased by endogenous AhR agonists present in culture medium. Th17 development from wild-type mice is suboptimal in the presence of the AhR antagonist CH-223191, similar to the situation in AhR-deficient mice, which show attenuated IL-17 production and no IL-22 production. The presence of natural AhR agonists in culture medium is also revealed by the induction of CYP1A1, a downstream target of AhR activation. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells. The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation. Our data emphasize that AhR activation plays an essential part in the development of Th17 cells and provide a rational explanation for the poor in vitro polarization of Th17 cells that is reported in the majority of publications for both mouse and human cells.

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Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from individual experiments with CD4 T cells from B6 (filled triangles) or AhR-deficient mice (open circles) cultured under Th17-polarizing conditions in IMDM or RPMI in the presence or absence of AhR antagonist (left). P value control versus AhR antagonist for B6 are <0.001; between IMDM and RPMI B6 control values P < 0.001. Data from four independent experiments are shown. (right) Percentage of IFN-γ producers (filled diamonds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 T cells purified from PBMCs of human blood and stimulated under Th17 conditions in IMDM (left) or RPMI (right) medium in the absence (top) or presence (bottom) of AhR antagonist. IL-17 versus IFN-γ intracellular staining is shown in the dot plots. The scatter plot shows the percentage of IL-17 producers from three different individuals (representing three independent experiments) obtained in IMDM versus RPMI medium.
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fig3: Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from individual experiments with CD4 T cells from B6 (filled triangles) or AhR-deficient mice (open circles) cultured under Th17-polarizing conditions in IMDM or RPMI in the presence or absence of AhR antagonist (left). P value control versus AhR antagonist for B6 are <0.001; between IMDM and RPMI B6 control values P < 0.001. Data from four independent experiments are shown. (right) Percentage of IFN-γ producers (filled diamonds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 T cells purified from PBMCs of human blood and stimulated under Th17 conditions in IMDM (left) or RPMI (right) medium in the absence (top) or presence (bottom) of AhR antagonist. IL-17 versus IFN-γ intracellular staining is shown in the dot plots. The scatter plot shows the percentage of IL-17 producers from three different individuals (representing three independent experiments) obtained in IMDM versus RPMI medium.

Mentions: Th17 differentiation in RPMI medium was consistently lower and similar to that of AhR-deficient cells that show attenuated Th17 polarization irrespective of the source of culture medium (Fig. 3 A). The AhR antagonist CH-223191 strongly reduced Th17 development from B6 CD4 T cells in IMDM, but even in RPMI medium, antagonizing AhR resulted in a further reduction of Th17 differentiation. In contrast to its effect on development of Th17 cells, Th1 polarization was not affected by the choice of culture medium or the presence of AhR antagonist. The development of iT reg cells was even increased in RPMI medium. Human Th17 cells also express AhR (3), and, similar to mouse T cells, the expansion of human Th17 cells from total CD4 T cells in peripheral blood was markedly better in IMDM compared with RPMI medium and strongly reduced in the presence of the AhR antagonist (Fig. 3 B).


Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells.

Veldhoen M, Hirota K, Christensen J, O'Garra A, Stockinger B - J. Exp. Med. (2008)

Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from individual experiments with CD4 T cells from B6 (filled triangles) or AhR-deficient mice (open circles) cultured under Th17-polarizing conditions in IMDM or RPMI in the presence or absence of AhR antagonist (left). P value control versus AhR antagonist for B6 are <0.001; between IMDM and RPMI B6 control values P < 0.001. Data from four independent experiments are shown. (right) Percentage of IFN-γ producers (filled diamonds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 T cells purified from PBMCs of human blood and stimulated under Th17 conditions in IMDM (left) or RPMI (right) medium in the absence (top) or presence (bottom) of AhR antagonist. IL-17 versus IFN-γ intracellular staining is shown in the dot plots. The scatter plot shows the percentage of IL-17 producers from three different individuals (representing three independent experiments) obtained in IMDM versus RPMI medium.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626686&req=5

fig3: Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from individual experiments with CD4 T cells from B6 (filled triangles) or AhR-deficient mice (open circles) cultured under Th17-polarizing conditions in IMDM or RPMI in the presence or absence of AhR antagonist (left). P value control versus AhR antagonist for B6 are <0.001; between IMDM and RPMI B6 control values P < 0.001. Data from four independent experiments are shown. (right) Percentage of IFN-γ producers (filled diamonds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 T cells purified from PBMCs of human blood and stimulated under Th17 conditions in IMDM (left) or RPMI (right) medium in the absence (top) or presence (bottom) of AhR antagonist. IL-17 versus IFN-γ intracellular staining is shown in the dot plots. The scatter plot shows the percentage of IL-17 producers from three different individuals (representing three independent experiments) obtained in IMDM versus RPMI medium.
Mentions: Th17 differentiation in RPMI medium was consistently lower and similar to that of AhR-deficient cells that show attenuated Th17 polarization irrespective of the source of culture medium (Fig. 3 A). The AhR antagonist CH-223191 strongly reduced Th17 development from B6 CD4 T cells in IMDM, but even in RPMI medium, antagonizing AhR resulted in a further reduction of Th17 differentiation. In contrast to its effect on development of Th17 cells, Th1 polarization was not affected by the choice of culture medium or the presence of AhR antagonist. The development of iT reg cells was even increased in RPMI medium. Human Th17 cells also express AhR (3), and, similar to mouse T cells, the expansion of human Th17 cells from total CD4 T cells in peripheral blood was markedly better in IMDM compared with RPMI medium and strongly reduced in the presence of the AhR antagonist (Fig. 3 B).

Bottom Line: Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR).However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells.The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK.

ABSTRACT
Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR). In this study, we show that differentiation of Th17 cells, but not Th1 or induced regulatory T (iT reg) cells, is increased by endogenous AhR agonists present in culture medium. Th17 development from wild-type mice is suboptimal in the presence of the AhR antagonist CH-223191, similar to the situation in AhR-deficient mice, which show attenuated IL-17 production and no IL-22 production. The presence of natural AhR agonists in culture medium is also revealed by the induction of CYP1A1, a downstream target of AhR activation. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells. The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation. Our data emphasize that AhR activation plays an essential part in the development of Th17 cells and provide a rational explanation for the poor in vitro polarization of Th17 cells that is reported in the majority of publications for both mouse and human cells.

Show MeSH
Related in: MedlinePlus