Synergistic effect of TGF-beta superfamily members on the induction of Foxp3+ Treg

Abstract

TGF-beta plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF-beta superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF-beta have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP-2 and -4, are unable to induce non-regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-beta to induce naïve CD4+CD25- cells to express Foxp3; however, BMP-2/4 and TGF-beta have a synergistic effect on the induction of Foxp3+ Treg. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF-beta. Data further advocate that TGF-beta is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP-2/4 and TGF-beta indicates that the simultaneous manipulation of TGF-beta and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.

Figure 1

BMP-2 and BMP-4 fail to induce Foxp3 expression on CD4+ cells. (A) Splenic naive CD4+CD25- cells isolated from C57BL/6 mice were stimulated with anti-CD3/CD28 coated beads (1:5) in the presence of IL-2 (20 u/ml) and TGF-β (2 ng/ml), or BMP-2 (10 ng/ml), or BMP-4 (10 ng/ml) for 4 days. Intracellular Foxp3 protein expression was analyzed by flow cytometry. Data show mean ± SEM of five independent experiments. (B) Representative histogram of experiments described in panel (A). The percentage of CD25+ cells expressing Foxp3 is shown in the upper right of the histogram. (C) Foxp3 mRNA expression as determined by real time PCR after normalization of β-actin. Data show mean ± SEM of four independent experiments. (D) Foxp3 and CD25 expression over time using a similar experimental design as in A. (E) Dose effect of BMP-2 and BMP-4 on the induction of Foxp3 expression. Data from panel D and E show mean ± SEM of four independent experiments.

Figure 2

BMP-2/4-primed CD4+ cells are unable to display the phenotypes and function of suppressor cells. (A) BMP2/4- or TGF-β-primed CD4+ cells were induced as described in Fig. 1. These cells were restimulated with anti-CD3 (0.25 μg/ml) in the presence of APC (3000-rad γ irradiation) for three days and proliferation was assayed by ³H-thymidine incorporation. (B) CD4+CD25- T cells labeled with CFSE were stimulated with soluble anti-CD3 and APC, or with BMP-2-, BMP-4-, TGF-β-primed CD4+ or control (Med) cells for 4 days. The ratio of conditioned cells to responder T cells is1:4. The % suppression was calculated as previously reported [11]. (C). Cytometric data representative of four independent experiments in panel (B). (D & F). C57BL/6 mice received intraperitoneal injections of BMP-2 (0.5 mg/mouse), BMP-4 (0.5 mg/mouse) or control PBS every other day for 10 days. The frequency (D) and suppressive activities (E) of CD4+CD25+Foxp3+ cells in thymus, spleen and blood in these mice were determined at day 12 after administration using flow cytometry and a standard in vitro suppressive assay as above. Each group includes 4 mice and values indicate mean ± SEM. Note there is no significant difference between mice receiving BMP-2/4 or PBS. (F) Naïve CD4+ cells isolated from C57BL/6 mice were treated with BMP-2/4 with or without TGF-β for 2 hours and the Smad1 and phosphorylated Smad1 expression was determined by western blot. Data are representative of three independent experiments.

Figure 2

BMP-2/4-primed CD4+ cells are unable to display the phenotypes and function of suppressor cells. (A) BMP2/4- or TGF-β-primed CD4+ cells were induced as described in Fig. 1. These cells were restimulated with anti-CD3 (0.25 μg/ml) in the presence of APC (3000-rad γ irradiation) for three days and proliferation was assayed by ³H-thymidine incorporation. (B) CD4+CD25- T cells labeled with CFSE were stimulated with soluble anti-CD3 and APC, or with BMP-2-, BMP-4-, TGF-β-primed CD4+ or control (Med) cells for 4 days. The ratio of conditioned cells to responder T cells is1:4. The % suppression was calculated as previously reported [11]. (C). Cytometric data representative of four independent experiments in panel (B). (D & F). C57BL/6 mice received intraperitoneal injections of BMP-2 (0.5 mg/mouse), BMP-4 (0.5 mg/mouse) or control PBS every other day for 10 days. The frequency (D) and suppressive activities (E) of CD4+CD25+Foxp3+ cells in thymus, spleen and blood in these mice were determined at day 12 after administration using flow cytometry and a standard in vitro suppressive assay as above. Each group includes 4 mice and values indicate mean ± SEM. Note there is no significant difference between mice receiving BMP-2/4 or PBS. (F) Naïve CD4+ cells isolated from C57BL/6 mice were treated with BMP-2/4 with or without TGF-β for 2 hours and the Smad1 and phosphorylated Smad1 expression was determined by western blot. Data are representative of three independent experiments.

Figure 3

Induction of Foxp3+ by TGF-β is independent upon BMP receptor signal. (A) Foxp3 expression was determined in CD4+ cells treated with medium, TGF-β, and both TGF-β and Noggin treated CD4+ cells. Data are representative of three independent experiments. (B) The suppressive activity of these cells was analyzed by a standard suppressive assay as described in Fig 1B. (C) The effects of Noggin on the Smad1 activation of BMP-4 on CD4+ cells. Naïve CD4+ cells were stimulated with BMP-4 with or without Noggin for 2 hours and Smad1 and phosphorylated Smad1 expression were determined by western blot. Experiments were repeated twice with similar results.

Figure 4

BMP-2/4 enhances TGF-β induced development of CD4+Foxp3+ Treg. (A) Effect of BMP-2/4 on the Foxp3 expression on CD4+ cells. Suppressive activities of Treg subsets against T cell proliferation in vitro (B) and anti-dsDNA production in cGVHD with a lupus syndrome model (n=4 mice each group) (C) were determined described the in the Materials and methods. Values are the mean ± SEM of three separate experiments (B). (D) The frequency of CD4+CD25+Foxp3+ cells in spleen, LN and gut of mice receiving PBS, or BMP-4, or TsA, or BMP-4+TsA at day 12 after treatment. Values indicate Mean ± SEM (left panel) of three mice in each group and representative of these experiments (right panel). Experiment was repeated with similar results. (E) Splenic CD4+CD25+ cells were sorted by FACSVantage and their suppressive activity against T cell proliferation in vitro was analyzed as panel A. (F and G). CD4+Foxp3+ cells were induced for 2 days as in Fig. 1 and labeled with CFSE, and stimulated with either IL-2 (40 units/ml), or BMP-2 (10 ng/ml), or BMP-4 (10 ng/ml) for additional 4 days. Total Foxp3+ cells were counted every two days (F) and Foxp3+ cell proliferation levels were analyzed by the dilution of CFSE dye on day 6 (G). Values indicate Mean ± SEM of three separate experiments (F) and the data presented are representative of these experiments (G). (H) Bcl-2 mRNA expression was determined by quantitative RT-PCR. Data show mean ± SEM of triplicate samples and experiment was performed with similar results.

Figure 4

BMP-2/4 enhances TGF-β induced development of CD4+Foxp3+ Treg. (A) Effect of BMP-2/4 on the Foxp3 expression on CD4+ cells. Suppressive activities of Treg subsets against T cell proliferation in vitro (B) and anti-dsDNA production in cGVHD with a lupus syndrome model (n=4 mice each group) (C) were determined described the in the Materials and methods. Values are the mean ± SEM of three separate experiments (B). (D) The frequency of CD4+CD25+Foxp3+ cells in spleen, LN and gut of mice receiving PBS, or BMP-4, or TsA, or BMP-4+TsA at day 12 after treatment. Values indicate Mean ± SEM (left panel) of three mice in each group and representative of these experiments (right panel). Experiment was repeated with similar results. (E) Splenic CD4+CD25+ cells were sorted by FACSVantage and their suppressive activity against T cell proliferation in vitro was analyzed as panel A. (F and G). CD4+Foxp3+ cells were induced for 2 days as in Fig. 1 and labeled with CFSE, and stimulated with either IL-2 (40 units/ml), or BMP-2 (10 ng/ml), or BMP-4 (10 ng/ml) for additional 4 days. Total Foxp3+ cells were counted every two days (F) and Foxp3+ cell proliferation levels were analyzed by the dilution of CFSE dye on day 6 (G). Values indicate Mean ± SEM of three separate experiments (F) and the data presented are representative of these experiments (G). (H) Bcl-2 mRNA expression was determined by quantitative RT-PCR. Data show mean ± SEM of triplicate samples and experiment was performed with similar results.

Figure 4

BMP-2/4 enhances TGF-β induced development of CD4+Foxp3+ Treg. (A) Effect of BMP-2/4 on the Foxp3 expression on CD4+ cells. Suppressive activities of Treg subsets against T cell proliferation in vitro (B) and anti-dsDNA production in cGVHD with a lupus syndrome model (n=4 mice each group) (C) were determined described the in the Materials and methods. Values are the mean ± SEM of three separate experiments (B). (D) The frequency of CD4+CD25+Foxp3+ cells in spleen, LN and gut of mice receiving PBS, or BMP-4, or TsA, or BMP-4+TsA at day 12 after treatment. Values indicate Mean ± SEM (left panel) of three mice in each group and representative of these experiments (right panel). Experiment was repeated with similar results. (E) Splenic CD4+CD25+ cells were sorted by FACSVantage and their suppressive activity against T cell proliferation in vitro was analyzed as panel A. (F and G). CD4+Foxp3+ cells were induced for 2 days as in Fig. 1 and labeled with CFSE, and stimulated with either IL-2 (40 units/ml), or BMP-2 (10 ng/ml), or BMP-4 (10 ng/ml) for additional 4 days. Total Foxp3+ cells were counted every two days (F) and Foxp3+ cell proliferation levels were analyzed by the dilution of CFSE dye on day 6 (G). Values indicate Mean ± SEM of three separate experiments (F) and the data presented are representative of these experiments (G). (H) Bcl-2 mRNA expression was determined by quantitative RT-PCR. Data show mean ± SEM of triplicate samples and experiment was performed with similar results.

Figure 5

Noggin blocks the BMP2/4 enhancement of TGF-β induced Foxp3 expression. Naïve CD4+CD25- cells isolated from C57BL/6 wild type and TGF-β RII knock out mice were stimulated as described in Fig. 1, in some cultures, Noggin (100 ng/ml) or ALK5 (TGF-β RI) inhibitor (5 μM) or DMSO control was added to cultures and Foxp3 expression was determined as above. Values indicate the mean ± SEM three independent experiments.

Figure 6

BMP2/4 on enhancement of TGF-β-induced CD4+Foxp3+ Treg development involves MAPK activation. (A) Naïve CD4+ cells were TCR (anti-CD3/CD28 coated bead 1:5) stimulated with or without BMP-4 ± TGF-β for 16 hours. Activated Smad2/3 expression was determined by western blot. These cells were stimulated with TCR in the presence of BMP-4, TGF-β or both (BMP-4+TGF-β) in various time-points as indicated and the levels of activated JNK (B) and ERK (C) were determined by western blot. (D) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4 + TGF-β in the presence or absence of MAPK inhibitors. (E) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4 ± TGF-β in ERK1 KO, JNK2 KO and littermate control mice for 4 days. (F) Foxp3 expression on CD4+ cells stimulated with TCR with BMP-2/4 ± TGF-β in IL-10 KO and littermate control mice for 4 days. In some experiments with wild type control mice, anti-IL-10, anti-IL-10R or control IgG were added to cultures. Data show mean ± SEM of three separate experiments or represent at least three independent experiments.

Figure 6

BMP2/4 on enhancement of TGF-β-induced CD4+Foxp3+ Treg development involves MAPK activation. (A) Naïve CD4+ cells were TCR (anti-CD3/CD28 coated bead 1:5) stimulated with or without BMP-4 ± TGF-β for 16 hours. Activated Smad2/3 expression was determined by western blot. These cells were stimulated with TCR in the presence of BMP-4, TGF-β or both (BMP-4+TGF-β) in various time-points as indicated and the levels of activated JNK (B) and ERK (C) were determined by western blot. (D) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4 + TGF-β in the presence or absence of MAPK inhibitors. (E) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4 ± TGF-β in ERK1 KO, JNK2 KO and littermate control mice for 4 days. (F) Foxp3 expression on CD4+ cells stimulated with TCR with BMP-2/4 ± TGF-β in IL-10 KO and littermate control mice for 4 days. In some experiments with wild type control mice, anti-IL-10, anti-IL-10R or control IgG were added to cultures. Data show mean ± SEM of three separate experiments or represent at least three independent experiments.

Figure 6

BMP2/4 on enhancement of TGF-β-induced CD4+Foxp3+ Treg development involves MAPK activation. (A) Naïve CD4+ cells were TCR (anti-CD3/CD28 coated bead 1:5) stimulated with or without BMP-4 ± TGF-β for 16 hours. Activated Smad2/3 expression was determined by western blot. These cells were stimulated with TCR in the presence of BMP-4, TGF-β or both (BMP-4+TGF-β) in various time-points as indicated and the levels of activated JNK (B) and ERK (C) were determined by western blot. (D) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4 + TGF-β in the presence or absence of MAPK inhibitors. (E) Foxp3 expression by TCR-stimulated CD4+ cells cultured with BMP-4 ± TGF-β in ERK1 KO, JNK2 KO and littermate control mice for 4 days. (F) Foxp3 expression on CD4+ cells stimulated with TCR with BMP-2/4 ± TGF-β in IL-10 KO and littermate control mice for 4 days. In some experiments with wild type control mice, anti-IL-10, anti-IL-10R or control IgG were added to cultures. Data show mean ± SEM of three separate experiments or represent at least three independent experiments.