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Human CD4 memory T cells can become CD4+IL-9+ T cells.

Putheti P, Awasthi A, Popoola J, Gao W, Strom TB - PLoS ONE (2010)

Bottom Line: IL-4+TGF-beta induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4(+)CD25(-)CD45RO(+) T cells as compared to naïve CD4(+)CD25(-)CD45RA(+) T cells.In addition, as compared to pbCD3/sCD28 plus TGF-beta stimulation, IL-4+TGF-beta stimulated memory CD4(+)CD25(-)CD45RO(+) T cells expressed reduced FOXP3 protein.Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-beta stimulated resting memory CD4(+) T cells demonstrated that the addition of IL-1beta, IL-12, and IL-21 further enhance IL-9 production.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-beta directs mouse CD4(+)CD25(-)CD62L(+) T cells to commit to inflammatory IL-9 producing CD4(+) T cells.

Methodology/principal findings: Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-beta induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4(+)CD25(-)CD45RO(+) T cells as compared to naïve CD4(+)CD25(-)CD45RA(+) T cells. In addition, as compared to pbCD3/sCD28 plus TGF-beta stimulation, IL-4+TGF-beta stimulated memory CD4(+)CD25(-)CD45RO(+) T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4(+)IL-9(+) T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNgamma or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-beta stimulated resting memory CD4(+) T cells demonstrated that the addition of IL-1beta, IL-12, and IL-21 further enhance IL-9 production.

Conclusions/significance: Taken together these data show both the differences and similarities between mouse and human CD4(+)IL9(+) T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4(+) T cells to antigen.

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IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4+CD25−CD45RO+ T cells.Resting memory CD4+CD25−CD45RO+ T cells (2.0×105/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4+CD25−CD45RO+ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (n = 3). Data is expressed as the mean±SD.
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pone-0008706-g004: IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4+CD25−CD45RO+ T cells.Resting memory CD4+CD25−CD45RO+ T cells (2.0×105/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4+CD25−CD45RO+ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (n = 3). Data is expressed as the mean±SD.

Mentions: As IL-9 is a pro-inflammatory cytokine, we then hypothesized that cytokines IL-12, IL-1β, IL-6, and IL21, that induce inflammatory Th1 or Th17 cell polarization, would enhance IL-4+TGF-β mediated generation of CD4+IL-9+ T cells in presence of pbCD3/sCD28 activation. IL-6 did not influence IL-9 secretion. IL-1α and IL-1β induce IL-9 production by activated murine-CD4+ T cells or -mast cells [15], [20]. IL-1β also stimulates IL-9 production by human eosinophils [21]. Strikingly, we found that IL-1β, in the presence of IL-4+TGF-β, induced four fold more IL-9 secretion by IL-4+TGF-β+pbCD3/sCD28-activated resting memory CD4+ T cells as compared to control cultures lacking IL-1β (p = 0.0009) (Fig 4). As macrophages are the major producers of IL-1β, this suggests their plausible role in stimulation of human CD4+IL-9+ T cells. We examined if IL-1β alone or in combination with either IL-4 or TGF-β is sufficient to induce IL-9 expression. As analyzed by flow cytometry, IL-4 or TGF-β induced generation of 2% or 4% of CD4+IL-9+ T cells did not alter upon adding IL-1β to the cultures, and no IL-9 was expressed in the presence of IL-1β alone (data not shown). Albeit to a lesser effect, IL-12 or IL-21 also amplified IL-9 secretion by IL-4+TGF-β+pbCD3/sCD28-activated memory CD4+CD25−CD45RO+ T cells (p = 0.001 or p = 0.014). As IL-2 was robustly secreted by IL-4+TGF-β treated resting memory CD4+ T cells (Fig 3A), it is not totally unexpected that addition of IL-2 to cultures of resting memory CD4+CD25−CD45RO+ T cells activated with pbCD3/sCD28 and IL-4+TGF-β did not have any significant effect on IL-9 secretion (Fig 4). Addition of neutralizing anti-IL-2 mAb to cultures had lead to CD4+ T cell death (data not shown). This data suggests IL-2 is required to maintain viability of IL-9 producers and IL-2 may not directly trigger IL-9 expression. Over all, anti-inflammatory cytokines, IL-4 and TGF-β, induce IL-9 production, and pro-inflammatory cytokines, IL-1β, IL-12, and IL-21, enhance IL-9 production.


Human CD4 memory T cells can become CD4+IL-9+ T cells.

Putheti P, Awasthi A, Popoola J, Gao W, Strom TB - PLoS ONE (2010)

IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4+CD25−CD45RO+ T cells.Resting memory CD4+CD25−CD45RO+ T cells (2.0×105/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4+CD25−CD45RO+ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (n = 3). Data is expressed as the mean±SD.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806834&req=5

pone-0008706-g004: IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4+CD25−CD45RO+ T cells.Resting memory CD4+CD25−CD45RO+ T cells (2.0×105/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4+CD25−CD45RO+ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (n = 3). Data is expressed as the mean±SD.
Mentions: As IL-9 is a pro-inflammatory cytokine, we then hypothesized that cytokines IL-12, IL-1β, IL-6, and IL21, that induce inflammatory Th1 or Th17 cell polarization, would enhance IL-4+TGF-β mediated generation of CD4+IL-9+ T cells in presence of pbCD3/sCD28 activation. IL-6 did not influence IL-9 secretion. IL-1α and IL-1β induce IL-9 production by activated murine-CD4+ T cells or -mast cells [15], [20]. IL-1β also stimulates IL-9 production by human eosinophils [21]. Strikingly, we found that IL-1β, in the presence of IL-4+TGF-β, induced four fold more IL-9 secretion by IL-4+TGF-β+pbCD3/sCD28-activated resting memory CD4+ T cells as compared to control cultures lacking IL-1β (p = 0.0009) (Fig 4). As macrophages are the major producers of IL-1β, this suggests their plausible role in stimulation of human CD4+IL-9+ T cells. We examined if IL-1β alone or in combination with either IL-4 or TGF-β is sufficient to induce IL-9 expression. As analyzed by flow cytometry, IL-4 or TGF-β induced generation of 2% or 4% of CD4+IL-9+ T cells did not alter upon adding IL-1β to the cultures, and no IL-9 was expressed in the presence of IL-1β alone (data not shown). Albeit to a lesser effect, IL-12 or IL-21 also amplified IL-9 secretion by IL-4+TGF-β+pbCD3/sCD28-activated memory CD4+CD25−CD45RO+ T cells (p = 0.001 or p = 0.014). As IL-2 was robustly secreted by IL-4+TGF-β treated resting memory CD4+ T cells (Fig 3A), it is not totally unexpected that addition of IL-2 to cultures of resting memory CD4+CD25−CD45RO+ T cells activated with pbCD3/sCD28 and IL-4+TGF-β did not have any significant effect on IL-9 secretion (Fig 4). Addition of neutralizing anti-IL-2 mAb to cultures had lead to CD4+ T cell death (data not shown). This data suggests IL-2 is required to maintain viability of IL-9 producers and IL-2 may not directly trigger IL-9 expression. Over all, anti-inflammatory cytokines, IL-4 and TGF-β, induce IL-9 production, and pro-inflammatory cytokines, IL-1β, IL-12, and IL-21, enhance IL-9 production.

Bottom Line: IL-4+TGF-beta induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4(+)CD25(-)CD45RO(+) T cells as compared to naïve CD4(+)CD25(-)CD45RA(+) T cells.In addition, as compared to pbCD3/sCD28 plus TGF-beta stimulation, IL-4+TGF-beta stimulated memory CD4(+)CD25(-)CD45RO(+) T cells expressed reduced FOXP3 protein.Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-beta stimulated resting memory CD4(+) T cells demonstrated that the addition of IL-1beta, IL-12, and IL-21 further enhance IL-9 production.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-beta directs mouse CD4(+)CD25(-)CD62L(+) T cells to commit to inflammatory IL-9 producing CD4(+) T cells.

Methodology/principal findings: Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-beta induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4(+)CD25(-)CD45RO(+) T cells as compared to naïve CD4(+)CD25(-)CD45RA(+) T cells. In addition, as compared to pbCD3/sCD28 plus TGF-beta stimulation, IL-4+TGF-beta stimulated memory CD4(+)CD25(-)CD45RO(+) T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4(+)IL-9(+) T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNgamma or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-beta stimulated resting memory CD4(+) T cells demonstrated that the addition of IL-1beta, IL-12, and IL-21 further enhance IL-9 production.

Conclusions/significance: Taken together these data show both the differences and similarities between mouse and human CD4(+)IL9(+) T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4(+) T cells to antigen.

Show MeSH
Related in: MedlinePlus