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TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

Oida T, Weiner HL - PLoS ONE (2010)

Bottom Line: It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb.We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment.Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs.

Methodology/principal findings: We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells.

Conclusions/significance: Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

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Induction of surface LAP by TGF-β.(A) BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 without (upper panels) or with 10 ng/ml recombinant TGF-β (lower panels) for 2 days and rested for 2 days. The cells were surface stained with anti-LAP TW7-16B4 or TW7-20B9, or anti-latent TGF-β/pro-TGF-β TW7-28G11 using goat anti-mouse Ig-PE secondary antibody, then fixed, and intracellularly stained with anti-Foxp3-Alexa Fluor647. (B) BALB/c CD4+CD25− T cells were stimulated with/without TGF-β, and then surface stained with ACP-conjugated anti-LAP TW7-20B9 and GARP-PE, followed by intracellular staining with anti-Foxp3-Alexa Fluor488. Foxp3− and Foxp3+ cells populations were gated and plotted by LAP and GARP expression.
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pone-0015523-g004: Induction of surface LAP by TGF-β.(A) BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 without (upper panels) or with 10 ng/ml recombinant TGF-β (lower panels) for 2 days and rested for 2 days. The cells were surface stained with anti-LAP TW7-16B4 or TW7-20B9, or anti-latent TGF-β/pro-TGF-β TW7-28G11 using goat anti-mouse Ig-PE secondary antibody, then fixed, and intracellularly stained with anti-Foxp3-Alexa Fluor647. (B) BALB/c CD4+CD25− T cells were stimulated with/without TGF-β, and then surface stained with ACP-conjugated anti-LAP TW7-20B9 and GARP-PE, followed by intracellular staining with anti-Foxp3-Alexa Fluor488. Foxp3− and Foxp3+ cells populations were gated and plotted by LAP and GARP expression.

Mentions: TGF-β converts Foxp3− CD4 T cells into induced Foxp3+ Tregs (iTregs) [1]. To determine whether iTregs also express surface LAP, we stimulated mouse CD4+CD25− T cells in the presence or absence of recombinant TGF-β and checked for surface LAP expression. As expected ∼25% of CD4+CD25- T cells were converted to Foxp3+ iTregs in presence of TGF-β (Figure 4A). We found that these iTregs expressed surface LAP. Interestingly, the Foxp3−-remaining cells also became surface LAP+ cells following culture in the presence of TGF-β. GARP expression correlated with surface LAP expression on both Foxp3+ cells and Foxp3− cells (Figure 4B), suggesting that surface LAP is GARP-dependent not only on natural Tregs and iTregs cells but also on non-Tregs.


TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

Oida T, Weiner HL - PLoS ONE (2010)

Induction of surface LAP by TGF-β.(A) BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 without (upper panels) or with 10 ng/ml recombinant TGF-β (lower panels) for 2 days and rested for 2 days. The cells were surface stained with anti-LAP TW7-16B4 or TW7-20B9, or anti-latent TGF-β/pro-TGF-β TW7-28G11 using goat anti-mouse Ig-PE secondary antibody, then fixed, and intracellularly stained with anti-Foxp3-Alexa Fluor647. (B) BALB/c CD4+CD25− T cells were stimulated with/without TGF-β, and then surface stained with ACP-conjugated anti-LAP TW7-20B9 and GARP-PE, followed by intracellular staining with anti-Foxp3-Alexa Fluor488. Foxp3− and Foxp3+ cells populations were gated and plotted by LAP and GARP expression.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991360&req=5

pone-0015523-g004: Induction of surface LAP by TGF-β.(A) BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 without (upper panels) or with 10 ng/ml recombinant TGF-β (lower panels) for 2 days and rested for 2 days. The cells were surface stained with anti-LAP TW7-16B4 or TW7-20B9, or anti-latent TGF-β/pro-TGF-β TW7-28G11 using goat anti-mouse Ig-PE secondary antibody, then fixed, and intracellularly stained with anti-Foxp3-Alexa Fluor647. (B) BALB/c CD4+CD25− T cells were stimulated with/without TGF-β, and then surface stained with ACP-conjugated anti-LAP TW7-20B9 and GARP-PE, followed by intracellular staining with anti-Foxp3-Alexa Fluor488. Foxp3− and Foxp3+ cells populations were gated and plotted by LAP and GARP expression.
Mentions: TGF-β converts Foxp3− CD4 T cells into induced Foxp3+ Tregs (iTregs) [1]. To determine whether iTregs also express surface LAP, we stimulated mouse CD4+CD25− T cells in the presence or absence of recombinant TGF-β and checked for surface LAP expression. As expected ∼25% of CD4+CD25- T cells were converted to Foxp3+ iTregs in presence of TGF-β (Figure 4A). We found that these iTregs expressed surface LAP. Interestingly, the Foxp3−-remaining cells also became surface LAP+ cells following culture in the presence of TGF-β. GARP expression correlated with surface LAP expression on both Foxp3+ cells and Foxp3− cells (Figure 4B), suggesting that surface LAP is GARP-dependent not only on natural Tregs and iTregs cells but also on non-Tregs.

Bottom Line: It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb.We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment.Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs.

Methodology/principal findings: We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells.

Conclusions/significance: Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

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