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Exposure to inflammatory cytokines selectively limits GM-CSF production by induced T regulatory cells.

Reynolds BC, Turner DG, McPherson RC, Prendergast CT, Phelps RG, Turner NA, O'Connor RA, Anderton SM - Eur. J. Immunol. (2014)

Bottom Line: Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important.We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN-γ, GM-CSF and TNF-α) upon secondary TCR stimulation.Furthermore, we show that IL-6 and IL-27 individually, or IL-2 and TGF-β in combination, can mediate the selective loss of GM-CSF production by iTreg cells.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK.

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Proinflammatory cytokines can selectively impair the production of GM-CSF by iTreg cells. (A–D) Sorted (>99% Foxp3gfp+) iTreg cells were restimulated with plate-bound anti-CD3 and anti-CD28 (both 2 μg/mL) with the addition of IL-12 (25 ng/mL), IL-27 (10 ng/mL), IL-6 (30 ng/mL), or IL-1β (10 ng/mL) for 72 h, with brefeldin A, PMA, and ionomycin for the final 4 h of culture. Cytokine production was then assessed by intracellular staining. (A) Representative flow cytometry plots of cytokine production by iTreg cells gated on live CD4+ cells (for gating strategy, see Supporting Information Fig. 3). Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. (B–D) Summary data for the proportion of restimulated iTreg cells producing (B) IFN-γ, (C) GM-CSF, and (D) TNF-α are shown as mean + SEM of triplicates. (E–G) iTreg cells were restimulated in the presence or absence of additional IL-2 (100 U/mL) and/or TGF-β (5 ng/mL), then analyzed as above. Summary data show the proportion of iTreg cells producing (E) IFN-γ, (F) GM-CSF, and (G) TNF-α are shown as mean ± SEM of triplicates. All data are from one experiment representative of three independent experiments. (H) iTreg cells were stained at the end of the 5-day Foxp3-induction culture for the expression of gp130 and CD126 (open histograms). Filled histograms show isotype control staining. Naïve T cells were also stained.
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fig05: Proinflammatory cytokines can selectively impair the production of GM-CSF by iTreg cells. (A–D) Sorted (>99% Foxp3gfp+) iTreg cells were restimulated with plate-bound anti-CD3 and anti-CD28 (both 2 μg/mL) with the addition of IL-12 (25 ng/mL), IL-27 (10 ng/mL), IL-6 (30 ng/mL), or IL-1β (10 ng/mL) for 72 h, with brefeldin A, PMA, and ionomycin for the final 4 h of culture. Cytokine production was then assessed by intracellular staining. (A) Representative flow cytometry plots of cytokine production by iTreg cells gated on live CD4+ cells (for gating strategy, see Supporting Information Fig. 3). Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. (B–D) Summary data for the proportion of restimulated iTreg cells producing (B) IFN-γ, (C) GM-CSF, and (D) TNF-α are shown as mean + SEM of triplicates. (E–G) iTreg cells were restimulated in the presence or absence of additional IL-2 (100 U/mL) and/or TGF-β (5 ng/mL), then analyzed as above. Summary data show the proportion of iTreg cells producing (E) IFN-γ, (F) GM-CSF, and (G) TNF-α are shown as mean ± SEM of triplicates. All data are from one experiment representative of three independent experiments. (H) iTreg cells were stained at the end of the 5-day Foxp3-induction culture for the expression of gp130 and CD126 (open histograms). Filled histograms show isotype control staining. Naïve T cells were also stained.

Mentions: The results in Fig. 4H–J suggested that component(s) of the in vivo inflammatory milieu were capable of selectively degrading the ability of iTreg cells to produce GM-CSF while maintaining IFN-γ and TNF-α production. To understand whether inflammatory cytokine(s) might be responsible for this, we returned to the in vitro restimulation of iTreg cells either under “neutral” conditions, or in the presence of additional cytokines (Fig. 5).


Exposure to inflammatory cytokines selectively limits GM-CSF production by induced T regulatory cells.

Reynolds BC, Turner DG, McPherson RC, Prendergast CT, Phelps RG, Turner NA, O'Connor RA, Anderton SM - Eur. J. Immunol. (2014)

Proinflammatory cytokines can selectively impair the production of GM-CSF by iTreg cells. (A–D) Sorted (>99% Foxp3gfp+) iTreg cells were restimulated with plate-bound anti-CD3 and anti-CD28 (both 2 μg/mL) with the addition of IL-12 (25 ng/mL), IL-27 (10 ng/mL), IL-6 (30 ng/mL), or IL-1β (10 ng/mL) for 72 h, with brefeldin A, PMA, and ionomycin for the final 4 h of culture. Cytokine production was then assessed by intracellular staining. (A) Representative flow cytometry plots of cytokine production by iTreg cells gated on live CD4+ cells (for gating strategy, see Supporting Information Fig. 3). Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. (B–D) Summary data for the proportion of restimulated iTreg cells producing (B) IFN-γ, (C) GM-CSF, and (D) TNF-α are shown as mean + SEM of triplicates. (E–G) iTreg cells were restimulated in the presence or absence of additional IL-2 (100 U/mL) and/or TGF-β (5 ng/mL), then analyzed as above. Summary data show the proportion of iTreg cells producing (E) IFN-γ, (F) GM-CSF, and (G) TNF-α are shown as mean ± SEM of triplicates. All data are from one experiment representative of three independent experiments. (H) iTreg cells were stained at the end of the 5-day Foxp3-induction culture for the expression of gp130 and CD126 (open histograms). Filled histograms show isotype control staining. Naïve T cells were also stained.
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fig05: Proinflammatory cytokines can selectively impair the production of GM-CSF by iTreg cells. (A–D) Sorted (>99% Foxp3gfp+) iTreg cells were restimulated with plate-bound anti-CD3 and anti-CD28 (both 2 μg/mL) with the addition of IL-12 (25 ng/mL), IL-27 (10 ng/mL), IL-6 (30 ng/mL), or IL-1β (10 ng/mL) for 72 h, with brefeldin A, PMA, and ionomycin for the final 4 h of culture. Cytokine production was then assessed by intracellular staining. (A) Representative flow cytometry plots of cytokine production by iTreg cells gated on live CD4+ cells (for gating strategy, see Supporting Information Fig. 3). Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. (B–D) Summary data for the proportion of restimulated iTreg cells producing (B) IFN-γ, (C) GM-CSF, and (D) TNF-α are shown as mean + SEM of triplicates. (E–G) iTreg cells were restimulated in the presence or absence of additional IL-2 (100 U/mL) and/or TGF-β (5 ng/mL), then analyzed as above. Summary data show the proportion of iTreg cells producing (E) IFN-γ, (F) GM-CSF, and (G) TNF-α are shown as mean ± SEM of triplicates. All data are from one experiment representative of three independent experiments. (H) iTreg cells were stained at the end of the 5-day Foxp3-induction culture for the expression of gp130 and CD126 (open histograms). Filled histograms show isotype control staining. Naïve T cells were also stained.
Mentions: The results in Fig. 4H–J suggested that component(s) of the in vivo inflammatory milieu were capable of selectively degrading the ability of iTreg cells to produce GM-CSF while maintaining IFN-γ and TNF-α production. To understand whether inflammatory cytokine(s) might be responsible for this, we returned to the in vitro restimulation of iTreg cells either under “neutral” conditions, or in the presence of additional cytokines (Fig. 5).

Bottom Line: Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important.We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN-γ, GM-CSF and TNF-α) upon secondary TCR stimulation.Furthermore, we show that IL-6 and IL-27 individually, or IL-2 and TGF-β in combination, can mediate the selective loss of GM-CSF production by iTreg cells.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK.

Show MeSH
Related in: MedlinePlus