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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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Related in: MedlinePlus

Production of IFN-γ, GM-CSF, and TNF-α occurs during primary iTreg-cell generation. Naive CD4+Foxp3gfp− cells were cultured in triplicate for 5 days in iTreg-cell conditions (IL-2 and TGF-β with plate-bound anti-CD3 and anti-CD28). Cells and supernatants were sampled daily. (A) The percentage of cells expressing Foxp3 as determined by intracellular Foxp3 staining and flow cytometry is shown. (B–D) Supernatants were analyzed by ELISA for the presence of (B) IFN-γ, (C) GM-CSF, and (D) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments. (E–G) Flow cytometry at the end of iTreg-cell culture (day 5) showing intracellular staining for Foxp3 and (E) IFN-γ, (F) GM-CSF, and (G) TNF-α. Numbers on plots refer to percentage in each quadrant rounded to the nearest integer.
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fig02: Production of IFN-γ, GM-CSF, and TNF-α occurs during primary iTreg-cell generation. Naive CD4+Foxp3gfp− cells were cultured in triplicate for 5 days in iTreg-cell conditions (IL-2 and TGF-β with plate-bound anti-CD3 and anti-CD28). Cells and supernatants were sampled daily. (A) The percentage of cells expressing Foxp3 as determined by intracellular Foxp3 staining and flow cytometry is shown. (B–D) Supernatants were analyzed by ELISA for the presence of (B) IFN-γ, (C) GM-CSF, and (D) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments. (E–G) Flow cytometry at the end of iTreg-cell culture (day 5) showing intracellular staining for Foxp3 and (E) IFN-γ, (F) GM-CSF, and (G) TNF-α. Numbers on plots refer to percentage in each quadrant rounded to the nearest integer.

Mentions: Cytokine production by iTreg cells was investigated further during the initial Foxp3-induction culture. Of note, Foxp3-gfp expression consistently increased to over 90% within 72 h (Fig. 2A). At that time, cytokine production was low or undetectable, but rose markedly in cultures sampled at days 4 and 5 (Fig. 2B–D). This argues against the possibility that the sole source of IFN-γ, GM-CSF, and TNF-α was cells that had not gained Foxp3 expression. This was further shown by clear populations of cytokine+ Foxp3+ cells at the end of the 5-day culture (Fig. 2E–F). This was particularly the case for TNF-α (Fig. 2F).


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)

Production of IFN-γ, GM-CSF, and TNF-α occurs during primary iTreg-cell generation. Naive CD4+Foxp3gfp− cells were cultured in triplicate for 5 days in iTreg-cell conditions (IL-2 and TGF-β with plate-bound anti-CD3 and anti-CD28). Cells and supernatants were sampled daily. (A) The percentage of cells expressing Foxp3 as determined by intracellular Foxp3 staining and flow cytometry is shown. (B–D) Supernatants were analyzed by ELISA for the presence of (B) IFN-γ, (C) GM-CSF, and (D) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments. (E–G) Flow cytometry at the end of iTreg-cell culture (day 5) showing intracellular staining for Foxp3 and (E) IFN-γ, (F) GM-CSF, and (G) TNF-α. Numbers on plots refer to percentage in each quadrant rounded to the nearest integer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Production of IFN-γ, GM-CSF, and TNF-α occurs during primary iTreg-cell generation. Naive CD4+Foxp3gfp− cells were cultured in triplicate for 5 days in iTreg-cell conditions (IL-2 and TGF-β with plate-bound anti-CD3 and anti-CD28). Cells and supernatants were sampled daily. (A) The percentage of cells expressing Foxp3 as determined by intracellular Foxp3 staining and flow cytometry is shown. (B–D) Supernatants were analyzed by ELISA for the presence of (B) IFN-γ, (C) GM-CSF, and (D) TNF-α. Data are shown as mean ± SEM of triplicates from one experiment representative of three experiments. (E–G) Flow cytometry at the end of iTreg-cell culture (day 5) showing intracellular staining for Foxp3 and (E) IFN-γ, (F) GM-CSF, and (G) TNF-α. Numbers on plots refer to percentage in each quadrant rounded to the nearest integer.
Mentions: Cytokine production by iTreg cells was investigated further during the initial Foxp3-induction culture. Of note, Foxp3-gfp expression consistently increased to over 90% within 72 h (Fig. 2A). At that time, cytokine production was low or undetectable, but rose markedly in cultures sampled at days 4 and 5 (Fig. 2B–D). This argues against the possibility that the sole source of IFN-γ, GM-CSF, and TNF-α was cells that had not gained Foxp3 expression. This was further shown by clear populations of cytokine+ Foxp3+ cells at the end of the 5-day culture (Fig. 2E–F). This was particularly the case for TNF-α (Fig. 2F).

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