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WASP regulates suppressor activity of human and murine CD4(+)CD25(+)FOXP3(+) natural regulatory T cells.

Marangoni F, Trifari S, Scaramuzza S, Panaroni C, Martino S, Notarangelo LD, Baz Z, Metin A, Cattaneo F, Villa A, Aiuti A, Battaglia M, Roncarolo MG, Dupré L - J. Exp. Med. (2007)

Bottom Line: Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering.Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells.Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), 20132 Milan, Italy.

ABSTRACT
A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4(+)CD25(+)FOXP3(+) natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS(-/-) mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS(-/-) nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS(-/-) nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.

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WASP expression in murine nTreg cells. (a) WASP expression in CD4+CD25+, CD4+CD25−, and CD8+ T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP+ cells. (b) F-actin and WASP localization in murine splenic WT CD4+CD25− and CD4+CD25+ T cells upon stimulation. CD4+CD25− effector T cells (left) and CD4+CD25+ nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4+CD25+ nTreg cells and WT CD4+CD25− effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P < 0.05, Student's t test.
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fig1: WASP expression in murine nTreg cells. (a) WASP expression in CD4+CD25+, CD4+CD25−, and CD8+ T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP+ cells. (b) F-actin and WASP localization in murine splenic WT CD4+CD25− and CD4+CD25+ T cells upon stimulation. CD4+CD25− effector T cells (left) and CD4+CD25+ nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4+CD25+ nTreg cells and WT CD4+CD25− effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P < 0.05, Student's t test.

Mentions: To investigate whether WASP plays a role in the function of nTreg cells, we first analyzed WASP expression and localization in mouse WT nTreg cells. nTreg cells from the spleens of C57BL/6 mice express WASP at levels comparable to that of CD4+CD25− T cells, CD8+ T cells (Fig. 1 a), B cells, NK cells, and granulocytes (not depicted). We previously showed that WASP promotes actin polarization at the immunological synapse of effector T cells (38). Accordingly, after activation of WT CD4+CD25− effector T cells with WT APCs in the presence of anti-CD3 mAbs, F-actin and WASP polarized to the APC contact site in ∼50% of the conjugates (Fig. 1, b and c). In contrast, <10% of CD4+CD25+ WT nTreg cells polarized F-actin and WASP at the contact of APCs. In most nTreg cells, a ring-like distribution of F-actin and WASP beneath the plasma membrane was observed (Fig. 1 b). The reduced polarization of F-actin and WASP in nTreg cells was not due to their defective ability to form conjugates with APCs because similar proportions of nTreg cells and effector T cells were in contact with APCs (not depicted). These data indicate that murine nTreg cells express levels of WASP similar to those of CD4+CD25− effector T cells, but they do not efficiently polarize F-actin and WASP to the APC contact site upon TCR triggering, suggesting that WASP-dependent actin polarization at the immunological synapse does not occur in nTreg cells under these in vitro conditions.


WASP regulates suppressor activity of human and murine CD4(+)CD25(+)FOXP3(+) natural regulatory T cells.

Marangoni F, Trifari S, Scaramuzza S, Panaroni C, Martino S, Notarangelo LD, Baz Z, Metin A, Cattaneo F, Villa A, Aiuti A, Battaglia M, Roncarolo MG, Dupré L - J. Exp. Med. (2007)

WASP expression in murine nTreg cells. (a) WASP expression in CD4+CD25+, CD4+CD25−, and CD8+ T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP+ cells. (b) F-actin and WASP localization in murine splenic WT CD4+CD25− and CD4+CD25+ T cells upon stimulation. CD4+CD25− effector T cells (left) and CD4+CD25+ nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4+CD25+ nTreg cells and WT CD4+CD25− effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P < 0.05, Student's t test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2118740&req=5

fig1: WASP expression in murine nTreg cells. (a) WASP expression in CD4+CD25+, CD4+CD25−, and CD8+ T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP+ cells. (b) F-actin and WASP localization in murine splenic WT CD4+CD25− and CD4+CD25+ T cells upon stimulation. CD4+CD25− effector T cells (left) and CD4+CD25+ nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4+CD25+ nTreg cells and WT CD4+CD25− effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P < 0.05, Student's t test.
Mentions: To investigate whether WASP plays a role in the function of nTreg cells, we first analyzed WASP expression and localization in mouse WT nTreg cells. nTreg cells from the spleens of C57BL/6 mice express WASP at levels comparable to that of CD4+CD25− T cells, CD8+ T cells (Fig. 1 a), B cells, NK cells, and granulocytes (not depicted). We previously showed that WASP promotes actin polarization at the immunological synapse of effector T cells (38). Accordingly, after activation of WT CD4+CD25− effector T cells with WT APCs in the presence of anti-CD3 mAbs, F-actin and WASP polarized to the APC contact site in ∼50% of the conjugates (Fig. 1, b and c). In contrast, <10% of CD4+CD25+ WT nTreg cells polarized F-actin and WASP at the contact of APCs. In most nTreg cells, a ring-like distribution of F-actin and WASP beneath the plasma membrane was observed (Fig. 1 b). The reduced polarization of F-actin and WASP in nTreg cells was not due to their defective ability to form conjugates with APCs because similar proportions of nTreg cells and effector T cells were in contact with APCs (not depicted). These data indicate that murine nTreg cells express levels of WASP similar to those of CD4+CD25− effector T cells, but they do not efficiently polarize F-actin and WASP to the APC contact site upon TCR triggering, suggesting that WASP-dependent actin polarization at the immunological synapse does not occur in nTreg cells under these in vitro conditions.

Bottom Line: Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering.Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells.Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), 20132 Milan, Italy.

ABSTRACT
A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4(+)CD25(+)FOXP3(+) natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS(-/-) mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS(-/-) nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS(-/-) nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.

Show MeSH
Related in: MedlinePlus