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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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TGF-β-induced Treg cells produce pro-inflammatory cytokines IFN-γ, GM-CSF, and TNF-α. (A–E) iTreg cells were generated from CD4+Foxp3gfp− cells (from Foxp3LuciDTR-4 mice) by 5-day culture as described in the Materials and methods. (A) Foxp3gfp expression pre and postsort on day 5 is shown. Numbers represent the percentage of Foxp3+ cells. (B–E) iTreg cells were then restimulated for 72 h with plate-bound anti-CD3 and anti-CD28 prior to intracellular staining for Foxp3 and cytokines and analysis by flow cytometry. Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. Data shown are from a single experiment representative of three experiments performed. (F–H) Tg4.Foxp3LuciDTR-4 iTreg cells (sorted to >98% Foxp3gfp+ purity) were restimulated for 48 h in triplicate with the indicated concentrations of MBP(Ac1–9) in the presence of splenic APCs. Supernatants were analyzed by ELISA for production of (F) IFN-γ, (G) GM-CSF, and (H) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments.
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fig01: TGF-β-induced Treg cells produce pro-inflammatory cytokines IFN-γ, GM-CSF, and TNF-α. (A–E) iTreg cells were generated from CD4+Foxp3gfp− cells (from Foxp3LuciDTR-4 mice) by 5-day culture as described in the Materials and methods. (A) Foxp3gfp expression pre and postsort on day 5 is shown. Numbers represent the percentage of Foxp3+ cells. (B–E) iTreg cells were then restimulated for 72 h with plate-bound anti-CD3 and anti-CD28 prior to intracellular staining for Foxp3 and cytokines and analysis by flow cytometry. Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. Data shown are from a single experiment representative of three experiments performed. (F–H) Tg4.Foxp3LuciDTR-4 iTreg cells (sorted to >98% Foxp3gfp+ purity) were restimulated for 48 h in triplicate with the indicated concentrations of MBP(Ac1–9) in the presence of splenic APCs. Supernatants were analyzed by ELISA for production of (F) IFN-γ, (G) GM-CSF, and (H) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments.

Mentions: iTreg cells were generated by 5 day culture of naive (Foxp3-GFP− CD4+) T cells with anti-CD3, anti-CD28, TGF-β, and IL-2. Highly pure iTreg cells were isolated by FACS-sorting based on Foxp3-GFP expression, (Fig. 1A) and were then restimulated with plate-bound anti-CD3 and anti-CD28 for 72 h. We used cytokine bead arrays to screen for the presence of inflammatory cytokines in supernatants from these secondary cultures. The majority of cytokines tested (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, and IL-22) were undetectable (data not shown). However, IFN-γ, TNF-α, and GM-CSF were detectable. Intracellular cytokine staining of restimulated iTreg cells demonstrated the loss of Foxp3 expression, as previously described 16, and confirmed the presence of cells staining positive for IFN-γ, GM-CSF, and TNF-α (Fig.1B–E). The majority of cells were TNF-α+ (Fig. 1D). Production of IFN-γ and GM-CSF was more restricted, and approximately 25% of cells were double positive for IFN-γ and GM-CSF (Fig. 1E). While IFN-γ and GM-CSF staining was seen chiefly in cells that had lost Foxp3 expression, TNF-α was also seen in a clear population that had retained Foxp3.


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)

TGF-β-induced Treg cells produce pro-inflammatory cytokines IFN-γ, GM-CSF, and TNF-α. (A–E) iTreg cells were generated from CD4+Foxp3gfp− cells (from Foxp3LuciDTR-4 mice) by 5-day culture as described in the Materials and methods. (A) Foxp3gfp expression pre and postsort on day 5 is shown. Numbers represent the percentage of Foxp3+ cells. (B–E) iTreg cells were then restimulated for 72 h with plate-bound anti-CD3 and anti-CD28 prior to intracellular staining for Foxp3 and cytokines and analysis by flow cytometry. Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. Data shown are from a single experiment representative of three experiments performed. (F–H) Tg4.Foxp3LuciDTR-4 iTreg cells (sorted to >98% Foxp3gfp+ purity) were restimulated for 48 h in triplicate with the indicated concentrations of MBP(Ac1–9) in the presence of splenic APCs. Supernatants were analyzed by ELISA for production of (F) IFN-γ, (G) GM-CSF, and (H) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments.
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Related In: Results  -  Collection

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fig01: TGF-β-induced Treg cells produce pro-inflammatory cytokines IFN-γ, GM-CSF, and TNF-α. (A–E) iTreg cells were generated from CD4+Foxp3gfp− cells (from Foxp3LuciDTR-4 mice) by 5-day culture as described in the Materials and methods. (A) Foxp3gfp expression pre and postsort on day 5 is shown. Numbers represent the percentage of Foxp3+ cells. (B–E) iTreg cells were then restimulated for 72 h with plate-bound anti-CD3 and anti-CD28 prior to intracellular staining for Foxp3 and cytokines and analysis by flow cytometry. Numbers on plots refer to percentage in each quadrant, rounded to the nearest integer. Data shown are from a single experiment representative of three experiments performed. (F–H) Tg4.Foxp3LuciDTR-4 iTreg cells (sorted to >98% Foxp3gfp+ purity) were restimulated for 48 h in triplicate with the indicated concentrations of MBP(Ac1–9) in the presence of splenic APCs. Supernatants were analyzed by ELISA for production of (F) IFN-γ, (G) GM-CSF, and (H) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments.
Mentions: iTreg cells were generated by 5 day culture of naive (Foxp3-GFP− CD4+) T cells with anti-CD3, anti-CD28, TGF-β, and IL-2. Highly pure iTreg cells were isolated by FACS-sorting based on Foxp3-GFP expression, (Fig. 1A) and were then restimulated with plate-bound anti-CD3 and anti-CD28 for 72 h. We used cytokine bead arrays to screen for the presence of inflammatory cytokines in supernatants from these secondary cultures. The majority of cytokines tested (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, and IL-22) were undetectable (data not shown). However, IFN-γ, TNF-α, and GM-CSF were detectable. Intracellular cytokine staining of restimulated iTreg cells demonstrated the loss of Foxp3 expression, as previously described 16, and confirmed the presence of cells staining positive for IFN-γ, GM-CSF, and TNF-α (Fig.1B–E). The majority of cells were TNF-α+ (Fig. 1D). Production of IFN-γ and GM-CSF was more restricted, and approximately 25% of cells were double positive for IFN-γ and GM-CSF (Fig. 1E). While IFN-γ and GM-CSF staining was seen chiefly in cells that had lost Foxp3 expression, TNF-α was also seen in a clear population that had retained Foxp3.

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