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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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Surface LAP/TGF-β expression on mouse activated CD4 T cells.(A) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. The cells were surface stained with anti-LAP mAbs using PE-labeled secondary antibodies, then intracellularly stained with anti-Foxp3-Alexa Fluor647. Staining with representative clones, anti-LAP mAbs TW7-16B4 and TW7-20B9, and anti-latent TGF-β/pro-TGF-β mAb TW7-28G11, are shown. (B) Activated BALB/c CD4 T cells were stained with anti-LAP TW7-20B9 (surface) and anti-Foxp3 (intracellular) (left), with anti-GARP (surface) and anti-Foxp3 (intracellular), or anti-LAP TW7-20B9 (surface) and GARP (surface) (right).
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pone-0015523-g001: Surface LAP/TGF-β expression on mouse activated CD4 T cells.(A) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. The cells were surface stained with anti-LAP mAbs using PE-labeled secondary antibodies, then intracellularly stained with anti-Foxp3-Alexa Fluor647. Staining with representative clones, anti-LAP mAbs TW7-16B4 and TW7-20B9, and anti-latent TGF-β/pro-TGF-β mAb TW7-28G11, are shown. (B) Activated BALB/c CD4 T cells were stained with anti-LAP TW7-20B9 (surface) and anti-Foxp3 (intracellular) (left), with anti-GARP (surface) and anti-Foxp3 (intracellular), or anti-LAP TW7-20B9 (surface) and GARP (surface) (right).

Mentions: It has been reported that human FOXP3 Tregs express surface LAP after activation [7], [8] by a GARP-mediated anchoring mechanism [8], [9]. We tested our anti-LAP/TGF-β mAbs for their ability to stain pre-activated mouse CD4 T cells. CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. Following this, they were surface stained with anti-LAP/TGF-β mAbs using PE-labeled anti-mouse IgG1 (for IgG1 subtype clones) or anti-mouse Igκ secondary antibody (for non-IgG1 clones), then fixed and intracellularly stained with anti-Foxp3-Alexa Fluor647. Of the 36 potential anti-LAP/TGF-β candidate clones, 31 clones surface stained Foxp3+ cells. Three representative clones (TW7-16B4, TW7-20B9, and TW7-28G11) are shown in Figure 1A and all 36 clones are shown in Figure S6. It should be noted that 24 of the 26 clones which immunoprecipitated Flag-mLAP as described above stained Foxp3+ CD4 T cells with a similar pattern. Among them, TW7-16B4 produced the highest staining signal followed by TW7-20B9. An anti-pro-TGF-β/latent TGF-β clone, TW7-28G11, also stained Foxp3+ CD4 T cells (Figure 1A and Figure S6), suggesting that surface LAP exists as pro-TGF-β and/or latent TGF-β rather than free LAP without mature TGF-β. Surface LAP staining strongly correlated with GARP expression (Figure 1B), indicating that surface LAP on mouse Foxp3+ CD4 Tregs is also anchored by GARP as on human FOXP3+ CD4 Tregs.


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

Oida T, Weiner HL - PLoS ONE (2010)

Surface LAP/TGF-β expression on mouse activated CD4 T cells.(A) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. The cells were surface stained with anti-LAP mAbs using PE-labeled secondary antibodies, then intracellularly stained with anti-Foxp3-Alexa Fluor647. Staining with representative clones, anti-LAP mAbs TW7-16B4 and TW7-20B9, and anti-latent TGF-β/pro-TGF-β mAb TW7-28G11, are shown. (B) Activated BALB/c CD4 T cells were stained with anti-LAP TW7-20B9 (surface) and anti-Foxp3 (intracellular) (left), with anti-GARP (surface) and anti-Foxp3 (intracellular), or anti-LAP TW7-20B9 (surface) and GARP (surface) (right).
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Related In: Results  -  Collection

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pone-0015523-g001: Surface LAP/TGF-β expression on mouse activated CD4 T cells.(A) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. The cells were surface stained with anti-LAP mAbs using PE-labeled secondary antibodies, then intracellularly stained with anti-Foxp3-Alexa Fluor647. Staining with representative clones, anti-LAP mAbs TW7-16B4 and TW7-20B9, and anti-latent TGF-β/pro-TGF-β mAb TW7-28G11, are shown. (B) Activated BALB/c CD4 T cells were stained with anti-LAP TW7-20B9 (surface) and anti-Foxp3 (intracellular) (left), with anti-GARP (surface) and anti-Foxp3 (intracellular), or anti-LAP TW7-20B9 (surface) and GARP (surface) (right).
Mentions: It has been reported that human FOXP3 Tregs express surface LAP after activation [7], [8] by a GARP-mediated anchoring mechanism [8], [9]. We tested our anti-LAP/TGF-β mAbs for their ability to stain pre-activated mouse CD4 T cells. CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for 2 days and rested for 1 day. Following this, they were surface stained with anti-LAP/TGF-β mAbs using PE-labeled anti-mouse IgG1 (for IgG1 subtype clones) or anti-mouse Igκ secondary antibody (for non-IgG1 clones), then fixed and intracellularly stained with anti-Foxp3-Alexa Fluor647. Of the 36 potential anti-LAP/TGF-β candidate clones, 31 clones surface stained Foxp3+ cells. Three representative clones (TW7-16B4, TW7-20B9, and TW7-28G11) are shown in Figure 1A and all 36 clones are shown in Figure S6. It should be noted that 24 of the 26 clones which immunoprecipitated Flag-mLAP as described above stained Foxp3+ CD4 T cells with a similar pattern. Among them, TW7-16B4 produced the highest staining signal followed by TW7-20B9. An anti-pro-TGF-β/latent TGF-β clone, TW7-28G11, also stained Foxp3+ CD4 T cells (Figure 1A and Figure S6), suggesting that surface LAP exists as pro-TGF-β and/or latent TGF-β rather than free LAP without mature TGF-β. Surface LAP staining strongly correlated with GARP expression (Figure 1B), indicating that surface LAP on mouse Foxp3+ CD4 Tregs is also anchored by GARP as on human FOXP3+ CD4 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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