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. 2010 Nov 24;5(11):e15523.
doi: 10.1371/journal.pone.0015523.

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

Takatoku Oida  1 , Howard L Weiner
Affiliations

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

Takatoku Oida et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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).
Figure 2
Figure 2. Surface LAP/TGF-β expression on mouse unstimulated CD4 T cells and time course analysis.
(A) Freshly prepared BALB/c CD4 T cells were surface stained with PE-conjugated anti-LAP/TGF-β mAbs (TW7-16B4, TW7-20B9, or TW7-28G11), anti-CD25-FITC, anti-CD4-APC, and 7-AAD. CD4+7-AAD− cells were gated. (B) BALB/c CD4 T cells were stimulated with plate-bound anti-CD3/anti-CD28 for two days, and then split in 10% FBS-IMDM containing 100 U/ml IL-2. The cells were surface stained with PE-conjugated anti-LAP TW7-20B9 followed by anti-Foxp3-Alexa Fluor647 (intracellular staining) (upper panels), or with APC-conjugated anti-LAP TW7-20B9 and GARP-PE (lower panels).
Figure 3
Figure 3. Induction of surface LAP by Foxp3 transduction.
BALB/c CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 and retrovirally transduced with pMCs-Foxp3-IRES-GFP vector. The cells were re-stimulated with plate-bound anti-CD3/anti-CD28 for 14 hrs and transferred to uncoated wells. 2 days after re-stimulation, the cells were stained with anti-LAP TW7-16B4 or TW7-20B9 using anti-mouse IgG1-APC secondary antibody.
Figure 4
Figure 4. 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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