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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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CD4+CD25− T cells activated with IL-4+TGF-β express more IL-9 than Th1, Th2, Th17, or iTregs.CD4+CD25− T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β or Th1-, Th2-, Th17-, iTreg-polarizing condition for 96hrs. Cells were harvested, gated on CD4+ T cells, and were analyzed for IL-9+ cells by flow cytometry or were used to quantitate IL-9 transcripts by real-time PCR. Data is expressed as the mean±SD. (A) 2% of Th2 cells, 4% of iTregs, or 10% of cells treated with IL-4+TGF-β in combination were IL-9+, whereas Th1-, Th17-, or Th0-cells had negligible number of IL-9+ cells (n = 7); (B) Log-transformed ratios of IL-9 mRNA copies to 18S rRNA are shown. Cells treated with IL-4+TGF-β in combination had significantly higher levels of IL-9 mRNA as compared to polarized Th1-, Th2-, Th17-, iTreg-, or Th0-cells (n = 9).
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pone-0008706-g002: CD4+CD25− T cells activated with IL-4+TGF-β express more IL-9 than Th1, Th2, Th17, or iTregs.CD4+CD25− T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β or Th1-, Th2-, Th17-, iTreg-polarizing condition for 96hrs. Cells were harvested, gated on CD4+ T cells, and were analyzed for IL-9+ cells by flow cytometry or were used to quantitate IL-9 transcripts by real-time PCR. Data is expressed as the mean±SD. (A) 2% of Th2 cells, 4% of iTregs, or 10% of cells treated with IL-4+TGF-β in combination were IL-9+, whereas Th1-, Th17-, or Th0-cells had negligible number of IL-9+ cells (n = 7); (B) Log-transformed ratios of IL-9 mRNA copies to 18S rRNA are shown. Cells treated with IL-4+TGF-β in combination had significantly higher levels of IL-9 mRNA as compared to polarized Th1-, Th2-, Th17-, iTreg-, or Th0-cells (n = 9).

Mentions: IL-9 is produced by mouse and human Th2 cells [15], and by mouse nTregs [13]. We then examined for IL-9 production by human CD4+CD25− T cells cultured in-vitro in Th1-, Th2-, and Th17-cell polarizing conditions. Robust IL-9 expression was detected in IL-4+TGF-β treated CD4+CD25− T cells at both mRNA and protein levels as compared to Th0- (p = 0.0006 and p = 0.01), Th1- (p = 0.002 and p = 0.001), Th2- (p = 0.002 and p = 0.04), Th17- (p = 0.001 and p = 0.01) cells, and iTregs (p = 0.002 and p = 0.04) (Fig 2). To our knowledge, this is the first report showing IL-9 expression by human iTregs.


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)

CD4+CD25− T cells activated with IL-4+TGF-β express more IL-9 than Th1, Th2, Th17, or iTregs.CD4+CD25− T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β or Th1-, Th2-, Th17-, iTreg-polarizing condition for 96hrs. Cells were harvested, gated on CD4+ T cells, and were analyzed for IL-9+ cells by flow cytometry or were used to quantitate IL-9 transcripts by real-time PCR. Data is expressed as the mean±SD. (A) 2% of Th2 cells, 4% of iTregs, or 10% of cells treated with IL-4+TGF-β in combination were IL-9+, whereas Th1-, Th17-, or Th0-cells had negligible number of IL-9+ cells (n = 7); (B) Log-transformed ratios of IL-9 mRNA copies to 18S rRNA are shown. Cells treated with IL-4+TGF-β in combination had significantly higher levels of IL-9 mRNA as compared to polarized Th1-, Th2-, Th17-, iTreg-, or Th0-cells (n = 9).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806834&req=5

pone-0008706-g002: CD4+CD25− T cells activated with IL-4+TGF-β express more IL-9 than Th1, Th2, Th17, or iTregs.CD4+CD25− T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β or Th1-, Th2-, Th17-, iTreg-polarizing condition for 96hrs. Cells were harvested, gated on CD4+ T cells, and were analyzed for IL-9+ cells by flow cytometry or were used to quantitate IL-9 transcripts by real-time PCR. Data is expressed as the mean±SD. (A) 2% of Th2 cells, 4% of iTregs, or 10% of cells treated with IL-4+TGF-β in combination were IL-9+, whereas Th1-, Th17-, or Th0-cells had negligible number of IL-9+ cells (n = 7); (B) Log-transformed ratios of IL-9 mRNA copies to 18S rRNA are shown. Cells treated with IL-4+TGF-β in combination had significantly higher levels of IL-9 mRNA as compared to polarized Th1-, Th2-, Th17-, iTreg-, or Th0-cells (n = 9).
Mentions: IL-9 is produced by mouse and human Th2 cells [15], and by mouse nTregs [13]. We then examined for IL-9 production by human CD4+CD25− T cells cultured in-vitro in Th1-, Th2-, and Th17-cell polarizing conditions. Robust IL-9 expression was detected in IL-4+TGF-β treated CD4+CD25− T cells at both mRNA and protein levels as compared to Th0- (p = 0.0006 and p = 0.01), Th1- (p = 0.002 and p = 0.001), Th2- (p = 0.002 and p = 0.04), Th17- (p = 0.001 and p = 0.01) cells, and iTregs (p = 0.002 and p = 0.04) (Fig 2). To our knowledge, this is the first report showing IL-9 expression by human iTregs.

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