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The BMP Pathway Participates in Human Naive CD4+ T Cell Activation and Homeostasis.

Martínez VG, Sacedón R, Hidalgo L, Valencia J, Fernández-Sevilla LM, Hernández-López C, Vicente A, Varas A - PLoS ONE (2015)

Bottom Line: Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling.Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis.Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Complutense University, Madrid, Spain.

ABSTRACT
Bone Morphogenetic Proteins (BMPs) form a group of secreted factors that belongs to the TGF-β superfamily. Among different roles in a number of immune cell types, BMPs are known to regulate T cell development within the thymus, although the role of BMP signaling in human mature T cells remains elusive. In this study, we demonstrate that canonical BMP signaling is necessary during two critical events that regulate the size and function of human naive CD4+ T cell population: activation and homeostasis. Upon stimulation via TCR, naive CD4+ T cells upregulate the expression of BMP ligands triggering canonical BMP signaling in CD25+ cells. Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling. Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis. Moreover, upregulation of two critical receptors for T cell homeostasis, CXCR4 and CCR9, triggered by IL-7 is also abrogated in the absence of BMP signaling. Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application.

No MeSH data available.


Related in: MedlinePlus

Canonical BMP pathway inhibition impairs T cell proliferation.(A) BrdU incorporation after 4 days of TCR stimulation with the indicated treatments. Results are represented as absorbance relative to control (horizontal bar). Means ± SD of two independent experiments run in duplicates are shown (* p≤0.05; by t test). Control refers to no treatment for BMP2 and BMP4, unspecific immunoglobulins for BMPRIA-Fc and DMSO for DMH1. (B-D) After 4 days of TCR stimulation in the presence of DMSO or DMH1, the proliferation rate, measured by CFSE loss (B), Hoechst staining (C) and number of cells recovered (D), and the percentage of apoptotic/necrotic cells (E) were analyzed by flow cytometry. Bars represent means ± SD of five independent experiments (* p≤0.05; by t test). Cell counts were performed in duplicates and the means ± SD of at least 4 independent experiments are shown. Histograms in (C) and dot plots in (E) correspond to one representative experiment. (F) Cells were harvested at the indicated time points and stained for CD25, phosphorylated Smad-1/5/8 (pBR-Smad) and Hoechst. Cell populations were defined according to the expression of CD25 and pBR-Smad and the percentage of Hoechst positive cells was determined for each subset. Means ± SD of three independent experiments are shown (* p≤0.05; ** p≤0.01; by t test).
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pone.0131453.g003: Canonical BMP pathway inhibition impairs T cell proliferation.(A) BrdU incorporation after 4 days of TCR stimulation with the indicated treatments. Results are represented as absorbance relative to control (horizontal bar). Means ± SD of two independent experiments run in duplicates are shown (* p≤0.05; by t test). Control refers to no treatment for BMP2 and BMP4, unspecific immunoglobulins for BMPRIA-Fc and DMSO for DMH1. (B-D) After 4 days of TCR stimulation in the presence of DMSO or DMH1, the proliferation rate, measured by CFSE loss (B), Hoechst staining (C) and number of cells recovered (D), and the percentage of apoptotic/necrotic cells (E) were analyzed by flow cytometry. Bars represent means ± SD of five independent experiments (* p≤0.05; by t test). Cell counts were performed in duplicates and the means ± SD of at least 4 independent experiments are shown. Histograms in (C) and dot plots in (E) correspond to one representative experiment. (F) Cells were harvested at the indicated time points and stained for CD25, phosphorylated Smad-1/5/8 (pBR-Smad) and Hoechst. Cell populations were defined according to the expression of CD25 and pBR-Smad and the percentage of Hoechst positive cells was determined for each subset. Means ± SD of three independent experiments are shown (* p≤0.05; ** p≤0.01; by t test).

Mentions: As a first approach, we analyzed whether modifications of BMP signaling could affect the proliferation of T cells. BMP signaling was activated by addition of exogenous BMP2 and BMP4, the only two members of the BMP2/4 subgroup which has shown the highest affinity for BMPRIA [2]. In parallel, BMP signaling was inhibited by two means: blocking soluble BMPs by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera, and inhibiting BR-Smad phosphorylation by BMP receptors type I with the inhibitor DMH1 [26]. As shown in Fig 3A, the addition of BMP2 and BMP4 slightly increased BrdU incorporation at day 4 of culture, although no statistical significance was reached. On the contrary, and supporting an autocrine role for BMP signaling pathway, both BMPRIA-Fc and DMH1 negatively affected the proliferative response of T cells, being this effect greater with the BMP inhibitor DMH1 (Fig 3A). The impairment of proliferation induced by BMP signaling blockade was confirmed by CFSE loss, showing that the presence of the BMP inhibitor DMH1 reduced the proliferative response induced by anti-CD3/CD28 stimulation by nearly an 80% with the highest dose (Fig 3B). This effect was dose-dependent and mainly caused by G0/G1 arrest, since the percentage of cycling cells was reduced by more than half in DMH1-treated cells (Fig 3C). Accordingly, the effects on cell cycle progression induced by DMH1 correlated well with the number of cells recovered after stimulation, being these numbers markedly lower in DMH1-treated cultures compared to control cultures (Fig 3D). It must be noted that the inhibition of proliferation induced by DMH1 treatment was not caused by exacerbated apoptosis, neither by toxic effects, since no remarkable differences were observed when cell viability was analyzed (Fig 3E).


The BMP Pathway Participates in Human Naive CD4+ T Cell Activation and Homeostasis.

Martínez VG, Sacedón R, Hidalgo L, Valencia J, Fernández-Sevilla LM, Hernández-López C, Vicente A, Varas A - PLoS ONE (2015)

Canonical BMP pathway inhibition impairs T cell proliferation.(A) BrdU incorporation after 4 days of TCR stimulation with the indicated treatments. Results are represented as absorbance relative to control (horizontal bar). Means ± SD of two independent experiments run in duplicates are shown (* p≤0.05; by t test). Control refers to no treatment for BMP2 and BMP4, unspecific immunoglobulins for BMPRIA-Fc and DMSO for DMH1. (B-D) After 4 days of TCR stimulation in the presence of DMSO or DMH1, the proliferation rate, measured by CFSE loss (B), Hoechst staining (C) and number of cells recovered (D), and the percentage of apoptotic/necrotic cells (E) were analyzed by flow cytometry. Bars represent means ± SD of five independent experiments (* p≤0.05; by t test). Cell counts were performed in duplicates and the means ± SD of at least 4 independent experiments are shown. Histograms in (C) and dot plots in (E) correspond to one representative experiment. (F) Cells were harvested at the indicated time points and stained for CD25, phosphorylated Smad-1/5/8 (pBR-Smad) and Hoechst. Cell populations were defined according to the expression of CD25 and pBR-Smad and the percentage of Hoechst positive cells was determined for each subset. Means ± SD of three independent experiments are shown (* p≤0.05; ** p≤0.01; by t test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481406&req=5

pone.0131453.g003: Canonical BMP pathway inhibition impairs T cell proliferation.(A) BrdU incorporation after 4 days of TCR stimulation with the indicated treatments. Results are represented as absorbance relative to control (horizontal bar). Means ± SD of two independent experiments run in duplicates are shown (* p≤0.05; by t test). Control refers to no treatment for BMP2 and BMP4, unspecific immunoglobulins for BMPRIA-Fc and DMSO for DMH1. (B-D) After 4 days of TCR stimulation in the presence of DMSO or DMH1, the proliferation rate, measured by CFSE loss (B), Hoechst staining (C) and number of cells recovered (D), and the percentage of apoptotic/necrotic cells (E) were analyzed by flow cytometry. Bars represent means ± SD of five independent experiments (* p≤0.05; by t test). Cell counts were performed in duplicates and the means ± SD of at least 4 independent experiments are shown. Histograms in (C) and dot plots in (E) correspond to one representative experiment. (F) Cells were harvested at the indicated time points and stained for CD25, phosphorylated Smad-1/5/8 (pBR-Smad) and Hoechst. Cell populations were defined according to the expression of CD25 and pBR-Smad and the percentage of Hoechst positive cells was determined for each subset. Means ± SD of three independent experiments are shown (* p≤0.05; ** p≤0.01; by t test).
Mentions: As a first approach, we analyzed whether modifications of BMP signaling could affect the proliferation of T cells. BMP signaling was activated by addition of exogenous BMP2 and BMP4, the only two members of the BMP2/4 subgroup which has shown the highest affinity for BMPRIA [2]. In parallel, BMP signaling was inhibited by two means: blocking soluble BMPs by addition of the recombinant human BMPR-IA/ALK-3 Fc chimera, and inhibiting BR-Smad phosphorylation by BMP receptors type I with the inhibitor DMH1 [26]. As shown in Fig 3A, the addition of BMP2 and BMP4 slightly increased BrdU incorporation at day 4 of culture, although no statistical significance was reached. On the contrary, and supporting an autocrine role for BMP signaling pathway, both BMPRIA-Fc and DMH1 negatively affected the proliferative response of T cells, being this effect greater with the BMP inhibitor DMH1 (Fig 3A). The impairment of proliferation induced by BMP signaling blockade was confirmed by CFSE loss, showing that the presence of the BMP inhibitor DMH1 reduced the proliferative response induced by anti-CD3/CD28 stimulation by nearly an 80% with the highest dose (Fig 3B). This effect was dose-dependent and mainly caused by G0/G1 arrest, since the percentage of cycling cells was reduced by more than half in DMH1-treated cells (Fig 3C). Accordingly, the effects on cell cycle progression induced by DMH1 correlated well with the number of cells recovered after stimulation, being these numbers markedly lower in DMH1-treated cultures compared to control cultures (Fig 3D). It must be noted that the inhibition of proliferation induced by DMH1 treatment was not caused by exacerbated apoptosis, neither by toxic effects, since no remarkable differences were observed when cell viability was analyzed (Fig 3E).

Bottom Line: Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling.Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis.Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Complutense University, Madrid, Spain.

ABSTRACT
Bone Morphogenetic Proteins (BMPs) form a group of secreted factors that belongs to the TGF-β superfamily. Among different roles in a number of immune cell types, BMPs are known to regulate T cell development within the thymus, although the role of BMP signaling in human mature T cells remains elusive. In this study, we demonstrate that canonical BMP signaling is necessary during two critical events that regulate the size and function of human naive CD4+ T cell population: activation and homeostasis. Upon stimulation via TCR, naive CD4+ T cells upregulate the expression of BMP ligands triggering canonical BMP signaling in CD25+ cells. Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling. Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis. Moreover, upregulation of two critical receptors for T cell homeostasis, CXCR4 and CCR9, triggered by IL-7 is also abrogated in the absence of BMP signaling. Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application.

No MeSH data available.


Related in: MedlinePlus