Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 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.

PubMed Disclaimer

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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rubtsov YP, Rudensky AY. TGFβ signalling in control of T-cell-mediated self-reactivity. Nat Rev Immunol. 2007;7:443–453. - PubMed
    1. Miyazono K, Ichijo H, Heldin CH. Transforming growth factor-β: latent forms, binding proteins and receptors. Growth Factors. 1993;8:11–22. - PubMed
    1. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor β. J Exp Med. 2001;194:629–644. - PMC - PubMed
    1. Oida T, Zhang X, Goto M, Hachimura S, Totsuka M, et al. CD4+CD25− T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-β-dependent mechanism. J Immunol. 2003;170:2516–2522. - PubMed
    1. Ochi H, Abraham M, Ishikawa H, Frenkel D, Yang K, et al. Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+CD25− LAP+ T cells. Nat Med. 2006;12:627–635. - PubMed

Publication types

MeSH terms