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Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9.

Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ - Diabetes (2009)

Bottom Line: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells.Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK. yunchuan.ding@nds.ox.ac.uk

ABSTRACT

Objective: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.

Research design and methods: We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.

Results: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.

Conclusions: We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

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T-cell blasts cocultured with MSCs exhibited hyporesponsiveness to exogenous IL-2 and lost surface expression of CD25. A: CFSE-labeled CD4+CD25− T-cells from naïve C57BL/6 mice (H2b) were cultured at a density of 2 × 105 per well in 96-well plates and stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (2 × 104). Exogenous IL-2 was added at concentrations of 15, 50, and 100 IU/ml. After 72 h, cell proliferation was analyzed by flow cytometry. Data are represented as means ± SD. Results are the average of three experiments of identical design. B: Bars show the SD CD4+CD25− T-cells were stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (MSC:T-cell ratio of 1:10) for 72 h. CD4+ T-cells were purified by MACs anti-CD4 beads and cultured with 50 IU/ml IL-2 for 72 h. T-cell proliferation was measured by 3H-Tdr incorporation added during the last 8 h of culture. C: T-cells stimulated with anti-CD3/CD28 beads in the presence or absence of MSC and harvested after 24 and 48 h. Surface expression of CD69, CD44, and CD25 was evaluated by flow cytometry. Histograms are representative of three independent experiments.
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Figure 2: T-cell blasts cocultured with MSCs exhibited hyporesponsiveness to exogenous IL-2 and lost surface expression of CD25. A: CFSE-labeled CD4+CD25− T-cells from naïve C57BL/6 mice (H2b) were cultured at a density of 2 × 105 per well in 96-well plates and stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (2 × 104). Exogenous IL-2 was added at concentrations of 15, 50, and 100 IU/ml. After 72 h, cell proliferation was analyzed by flow cytometry. Data are represented as means ± SD. Results are the average of three experiments of identical design. B: Bars show the SD CD4+CD25− T-cells were stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (MSC:T-cell ratio of 1:10) for 72 h. CD4+ T-cells were purified by MACs anti-CD4 beads and cultured with 50 IU/ml IL-2 for 72 h. T-cell proliferation was measured by 3H-Tdr incorporation added during the last 8 h of culture. C: T-cells stimulated with anti-CD3/CD28 beads in the presence or absence of MSC and harvested after 24 and 48 h. Surface expression of CD69, CD44, and CD25 was evaluated by flow cytometry. Histograms are representative of three independent experiments.

Mentions: In many situations of in vitro T-cell hyporesponsiveness, T-cell proliferation can be restored by the addition of exogenous interleukin (IL)-2 (24,25). Therefore, we asked whether the MSC-mediated suppressive activity on T-cell proliferation could be rescued by the addition of IL-2. Carboxyfluorescein succinimidyl ester (CFSE)-labeled T-cells were stimulated in the presence or absence of MSCs as in Fig. 1D and proliferation determined by CFSE dilution. As shown in Fig. 2A, in the absence of MSCs, virtually 90% of the T-cells proliferated, but this was reduced to ∼10% by the addition of MSCs. Surprisingly, proliferation was not restored by the addition of exogenous IL-2. We then evaluated whether stimulated T-cells might regain their responsiveness to IL-2 after MSCs were removed. Following stimulation with anti-CD3/CD28 beads in the presence or absence of MSCs, T-cells were reisolated by positive selection then incubated with exogenous IL-2. As shown in Fig. 2B, while cells stimulated in the primary culture without MSCs proliferated vigorously in response to IL-2 in the secondary cultures, cells stimulated initially in the presence of MSCs were unable to respond to exogenous IL-2. Thus, MSC-mediated IL-2 unresponsiveness appears to be a relatively stable phenomenon.


Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9.

Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ - Diabetes (2009)

T-cell blasts cocultured with MSCs exhibited hyporesponsiveness to exogenous IL-2 and lost surface expression of CD25. A: CFSE-labeled CD4+CD25− T-cells from naïve C57BL/6 mice (H2b) were cultured at a density of 2 × 105 per well in 96-well plates and stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (2 × 104). Exogenous IL-2 was added at concentrations of 15, 50, and 100 IU/ml. After 72 h, cell proliferation was analyzed by flow cytometry. Data are represented as means ± SD. Results are the average of three experiments of identical design. B: Bars show the SD CD4+CD25− T-cells were stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (MSC:T-cell ratio of 1:10) for 72 h. CD4+ T-cells were purified by MACs anti-CD4 beads and cultured with 50 IU/ml IL-2 for 72 h. T-cell proliferation was measured by 3H-Tdr incorporation added during the last 8 h of culture. C: T-cells stimulated with anti-CD3/CD28 beads in the presence or absence of MSC and harvested after 24 and 48 h. Surface expression of CD69, CD44, and CD25 was evaluated by flow cytometry. Histograms are representative of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 2: T-cell blasts cocultured with MSCs exhibited hyporesponsiveness to exogenous IL-2 and lost surface expression of CD25. A: CFSE-labeled CD4+CD25− T-cells from naïve C57BL/6 mice (H2b) were cultured at a density of 2 × 105 per well in 96-well plates and stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (2 × 104). Exogenous IL-2 was added at concentrations of 15, 50, and 100 IU/ml. After 72 h, cell proliferation was analyzed by flow cytometry. Data are represented as means ± SD. Results are the average of three experiments of identical design. B: Bars show the SD CD4+CD25− T-cells were stimulated with anti-CD3/CD28 beads alone or in the presence of MSCs (MSC:T-cell ratio of 1:10) for 72 h. CD4+ T-cells were purified by MACs anti-CD4 beads and cultured with 50 IU/ml IL-2 for 72 h. T-cell proliferation was measured by 3H-Tdr incorporation added during the last 8 h of culture. C: T-cells stimulated with anti-CD3/CD28 beads in the presence or absence of MSC and harvested after 24 and 48 h. Surface expression of CD69, CD44, and CD25 was evaluated by flow cytometry. Histograms are representative of three independent experiments.
Mentions: In many situations of in vitro T-cell hyporesponsiveness, T-cell proliferation can be restored by the addition of exogenous interleukin (IL)-2 (24,25). Therefore, we asked whether the MSC-mediated suppressive activity on T-cell proliferation could be rescued by the addition of IL-2. Carboxyfluorescein succinimidyl ester (CFSE)-labeled T-cells were stimulated in the presence or absence of MSCs as in Fig. 1D and proliferation determined by CFSE dilution. As shown in Fig. 2A, in the absence of MSCs, virtually 90% of the T-cells proliferated, but this was reduced to ∼10% by the addition of MSCs. Surprisingly, proliferation was not restored by the addition of exogenous IL-2. We then evaluated whether stimulated T-cells might regain their responsiveness to IL-2 after MSCs were removed. Following stimulation with anti-CD3/CD28 beads in the presence or absence of MSCs, T-cells were reisolated by positive selection then incubated with exogenous IL-2. As shown in Fig. 2B, while cells stimulated in the primary culture without MSCs proliferated vigorously in response to IL-2 in the secondary cultures, cells stimulated initially in the presence of MSCs were unable to respond to exogenous IL-2. Thus, MSC-mediated IL-2 unresponsiveness appears to be a relatively stable phenomenon.

Bottom Line: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells.Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK. yunchuan.ding@nds.ox.ac.uk

ABSTRACT

Objective: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.

Research design and methods: We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.

Results: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.

Conclusions: We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

Show MeSH
Related in: MedlinePlus