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. 2015 Jun 25;10(6):e0131453.
doi: 10.1371/journal.pone.0131453. eCollection 2015.

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

Víctor G Martínez  1 , Rosa Sacedón  1 , Laura Hidalgo  1 , Jaris Valencia  1 , Lidia M Fernández-Sevilla  1 , Carmen Hernández-López  1 , Angeles Vicente  1 , Alberto Varas  1
Affiliations

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

Víctor G Martínez et al. PLoS One. .

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.

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Conflict of interest statement

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

Figures

Fig 1
Fig 1. BMP signaling is activated by TCR stimulation in naive CD4+ T cells.
Freshly isolated human peripheral blood naive CD4+ T cells were stimulated with anti-CD3/CD28 mAb. (A) Transcripts for several components of the canonical BMP signaling pathway were determined by real-time PCR ex vivo (0h) or after 6 days of stimulation (6d). GNB2L1 was used as endogenous control. Means ± SD of at least three independent experiments run in duplicates are shown. Note the logarithmic scale on y-axis. (B) Percentage of BMPRIA+ cells detected by flow cytometry throughout the culture. Bars represent the mean ± SD of two to five independent experiments. (C) Expression of BMPRIA and CD25 in T cells (upper dot plots) and differential expression of BMPRIA in the CD25- and CD25+ cell populations (lower histograms) during activation. A representative experiment out of four is shown. (D) Expression of BMPRIA in T cells cultured with different stimuli. Grey histograms represent isotype controls. Similar stainings were obtained in two to three independent experiments. mDCs: mature dendritic cells; PHA: Phytohaemagglutinin; Ck: cytokine cocktail (rhIL-2, rhIL-6, rhTNF-α) (E) Determination of BMP2/4 and BMP6 production by flow cytometry in T cells cultured in media alone (grey histograms) or in the presence of anti-CD3/CD28 mAb (black histograms). Grey filled histograms represent isotype control stainings. A representative experiment out of three is shown. (F) Expression of CD25 and phosphorylated BR-Smad (pBR-Smads) during activation. For comparison, T cells were kept in culture media alone. Results are representative of three independent experiments.
Fig 2
Fig 2. BMPRIA expression during CD4+ T cell activation.
After 4 days of culture, TCR-activated T cells were stained for CD25 and BMPRIA and the CD25+BMPRIA- and CD25+BMPRIA+ cell populations were isolated by cell sorting. The sorted populations were then re-stimulated with anti-CD3/CD28 mAb and the expression of CD25 and BMPRIA was evaluated by flow cytometry after 36 hours of culture. A representative experiment out of two is shown.
Fig 3
Fig 3. 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).
Fig 4
Fig 4. DMH1 effects on IL-2 expression.
TCR-induced IL-2 production by T cells in the presence of DMSO or DMH1 at day 4 (A) and 2 (B, left graph). (B, right graph) mRNA expression for IL2 after 2 days of activation. GNB2L1 was used as endogenous control. Means ± SD of three to five independent experiments performed in duplicates are shown (* p≤0.05; ** p≤0.01; by t test). (C) Proliferation rate measured by CFSE loss in T cells after 4 days of TCR stimulation with DMSO, DMH1 alone or DMH1 supplemented with the indicated doses of rhIL-2. Bars represent the means ± SD of three independent experiments (* p≤0.05; by t test).
Fig 5
Fig 5. Canonical BMP pathway and IL-7 signaling.
(A) Expression of BMP2/4 and BMP6 was determined by flow cytometry at the indicated time points in T cells cultured in media alone (grey histograms) or in the presence of IL-7 (5 ng/ml) (black histograms). Grey filled histograms represent isotype control stainings. A representative experiment out of four is shown. (B) Differential expression of phosphorylated Smad-1/5/8 (pBR-Smad) analyzed by flow cytometry in naive CD4+ T cells after 11 days of culture in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM). Percentages represent the increment relative to cultures in media alone. One representative of three independent experiments is shown.
Fig 6
Fig 6. Effects of BMP pathway blockade on IL-7-induced T cell homeostasis.
(A) Naive CD4+ T cells cultured in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM) were harvested and counted at the indicated time points. Cell counts were performed in duplicates. Results represent the mean ± SD of four to twelve samples pooled from at least two independent experiments (** p≤0.01; *** p≤0.005; by t test. IL-7/DMSO vs IL-7/DMH1). (B) Differential expression of CD127 analyzed by flow cytometry after 36 hours of culture under the indicated conditions. Similar stainings were obtained in two independent experiments. (C) Proliferation rate measured by CFSE loss along 20 days in T cells cultured in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM). Means ± SD of four independent experiments are shown (* p≤0.05; by t test. IL-7/DMSO vs IL-7/DMH1). (D) Cell viability calculated as percentage of PI-/Annexin-V- cells throughout the culture. Means ± SD of four independent experiments are shown (** p≤0.01; *** p≤0.005; by t test. IL-7/DMSO vs IL-7/DMH1). (E) Bcl-2 levels determined by flow cytometry after 6 days of culture. White filled histograms represent media alone; grey-filled IL-7/DMSO; black-filled IL-7/DMH1. The mean fluorescence intensity is indicated in each histogram. Similar stainings were obtained in two independent experiments.
Fig 7
Fig 7. DMH1 effects on IL-7-induced homing receptor modulation.
T cells were cultured in media alone or supplemented with IL-7 and DMSO or IL-7 and DMH1 (40 μM) and the expression of several homing receptors (A), CXCR4 (B) and CCR9 (C) was analyzed by flow cytometry after 36 hours of culture. Bars represent the mean ± SD of two independent experiments (* p≤0.05; by t test. IL-7/DMSO vs IL-7/DMH1).
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This work was supported by grants SAF2012-33180 (Ministerio de Economía y Competitividad), S2010/BMD-2420 (Comunidad de Madrid) and RD12/0019/0007 (Instituto de Salud Carlos III). L.H. is supported by pre-doctoral fellowships (AP2009-4324) from the Ministerio de Educación, Cultura y Deporte.