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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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Related in: MedlinePlus

Cytokine and transcription factor profile of memory CD4+CD25−CD45RO+ T cells activated with IL-4+TGF-β.A) Log-transformed quantities of cytokines (pg/ml) are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4+ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4+CD25−CD45RO+ T cells not treated with IL-4+TGF-β (n = 3). Data is expressed as the mean±SD. B) Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (n = 3). As IL-9 is expressed in only 10% of all CD4+ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9+ cell. CD4+IL-9+ T cells expressed GATA3 and RORC, but not Tbet. C) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9+ or/and FOXP3+ cells were analyzed. 25% of CD4+IL-9+ T cells were also FOXP3+. Data are representative of seven independent experiments. D) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (n = 3). Data is expressed as the mean±SD.
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pone-0008706-g003: Cytokine and transcription factor profile of memory CD4+CD25−CD45RO+ T cells activated with IL-4+TGF-β.A) Log-transformed quantities of cytokines (pg/ml) are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4+ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4+CD25−CD45RO+ T cells not treated with IL-4+TGF-β (n = 3). Data is expressed as the mean±SD. B) Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (n = 3). As IL-9 is expressed in only 10% of all CD4+ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9+ cell. CD4+IL-9+ T cells expressed GATA3 and RORC, but not Tbet. C) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9+ or/and FOXP3+ cells were analyzed. 25% of CD4+IL-9+ T cells were also FOXP3+. Data are representative of seven independent experiments. D) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (n = 3). Data is expressed as the mean±SD.

Mentions: Next, we examined the cytokine profile of these IL-9 producing resting memory CD4+CD25−CD45RO+ T cells 96hrs after in-vitro stimulation with pbCD3/sCD28 in presence of IL-4+TGF-β. In this setting, activated memory CD4+CD25−CD45RO+ T cells secreted low levels of IFNγ (p = 0.004), IL-13 (p = 0.004), IL-17 (p = 0.004), IL-5 and IL-10 (not statistically significant) in comparison to control cultures lacking IL-4+TGF-β, as analyzed by ELISA (Fig 3A). Note that in the presence of IL-4+TGF-β and pbCD3/sCD28 stimulation, memory CD4+CD25−CD45RO+ T cells secreted higher levels of IL-2 as compared to control cultures lacking IL-4+TGF-β (p = 0.008). This suggests a possible role of IL-2 in maintaining viability of CD4+IL-9+T cells generated from resting memory CD4+CD25−CD45RO+ T cells. As IL-9 is expressed by human Th2 cells and mouse “Th9” cells produced IL-10, we hypothesized that TGF-β would synergize with IL-4 in inducing production of all the Th2 cytokines. We found that TGF-β synergized with IL-4 in elevating IL-9 production, but inhibited IL-4 mediated IL-5, IL-10, and IL-13 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)

Cytokine and transcription factor profile of memory CD4+CD25−CD45RO+ T cells activated with IL-4+TGF-β.A) Log-transformed quantities of cytokines (pg/ml) are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4+ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4+CD25−CD45RO+ T cells not treated with IL-4+TGF-β (n = 3). Data is expressed as the mean±SD. B) Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (n = 3). As IL-9 is expressed in only 10% of all CD4+ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9+ cell. CD4+IL-9+ T cells expressed GATA3 and RORC, but not Tbet. C) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9+ or/and FOXP3+ cells were analyzed. 25% of CD4+IL-9+ T cells were also FOXP3+. Data are representative of seven independent experiments. D) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (n = 3). Data is expressed as the mean±SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806834&req=5

pone-0008706-g003: Cytokine and transcription factor profile of memory CD4+CD25−CD45RO+ T cells activated with IL-4+TGF-β.A) Log-transformed quantities of cytokines (pg/ml) are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4+ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4+CD25−CD45RO+ T cells not treated with IL-4+TGF-β (n = 3). Data is expressed as the mean±SD. B) Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (n = 3). As IL-9 is expressed in only 10% of all CD4+ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9+ cell. CD4+IL-9+ T cells expressed GATA3 and RORC, but not Tbet. C) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9+ or/and FOXP3+ cells were analyzed. 25% of CD4+IL-9+ T cells were also FOXP3+. Data are representative of seven independent experiments. D) CD4+CD25−CD45RO+ T cells (1.0×106/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (n = 3). Data is expressed as the mean±SD.
Mentions: Next, we examined the cytokine profile of these IL-9 producing resting memory CD4+CD25−CD45RO+ T cells 96hrs after in-vitro stimulation with pbCD3/sCD28 in presence of IL-4+TGF-β. In this setting, activated memory CD4+CD25−CD45RO+ T cells secreted low levels of IFNγ (p = 0.004), IL-13 (p = 0.004), IL-17 (p = 0.004), IL-5 and IL-10 (not statistically significant) in comparison to control cultures lacking IL-4+TGF-β, as analyzed by ELISA (Fig 3A). Note that in the presence of IL-4+TGF-β and pbCD3/sCD28 stimulation, memory CD4+CD25−CD45RO+ T cells secreted higher levels of IL-2 as compared to control cultures lacking IL-4+TGF-β (p = 0.008). This suggests a possible role of IL-2 in maintaining viability of CD4+IL-9+T cells generated from resting memory CD4+CD25−CD45RO+ T cells. As IL-9 is expressed by human Th2 cells and mouse “Th9” cells produced IL-10, we hypothesized that TGF-β would synergize with IL-4 in inducing production of all the Th2 cytokines. We found that TGF-β synergized with IL-4 in elevating IL-9 production, but inhibited IL-4 mediated IL-5, IL-10, and IL-13 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