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Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis.

Schurgers E, Kelchtermans H, Mitera T, Geboes L, Matthys P - Arthritis Res. Ther. (2010)

Bottom Line: In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA.Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, Faculty of Medicine, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. evelien.schurgers@rega.kuleuven.be

ABSTRACT

Introduction: The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-gamma (IFN-gamma) in these processes.

Methods: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-gammaR KO MSC.

Results: Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-gammaR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.

Conclusions: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

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Mesenchymal stem cells (MSCs) inhibit the anti-CD3-induced proliferation of CD4+ T cells in vitro. (a) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or interferon-gamma receptor knockout (IFN-γR KO) MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of four cultures ± standard error of the mean (SEM). Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test). (b) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or IFN-γR KO MSCs for 72 hours. The proliferation of CD4+ T cells was analyzed by detection of CFSE dilution by flow cytometry. The percentage inhibition (100 × [(percentage of proliferating CD4+ cells not treated with MSCs -- percentage of proliferating CD4+ cells treated with MSCs)/percentage of proliferating CD4+ cells not treated with MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of three cultures ± SEM. Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test).
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Figure 2: Mesenchymal stem cells (MSCs) inhibit the anti-CD3-induced proliferation of CD4+ T cells in vitro. (a) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or interferon-gamma receptor knockout (IFN-γR KO) MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of four cultures ± standard error of the mean (SEM). Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test). (b) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or IFN-γR KO MSCs for 72 hours. The proliferation of CD4+ T cells was analyzed by detection of CFSE dilution by flow cytometry. The percentage inhibition (100 × [(percentage of proliferating CD4+ cells not treated with MSCs -- percentage of proliferating CD4+ cells treated with MSCs)/percentage of proliferating CD4+ cells not treated with MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of three cultures ± SEM. Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test).

Mentions: To investigate the immunosuppressive potential of MSCs in vitro, we tested their effect on the anti-CD3-induced proliferation of CD4+ T cells. T cells were stimulated in vitro with anti-CD3 antibody in the absence or presence of MSCs and their proliferation was analyzed by thymidine incorporation. MSCs of wild-type origin dose-dependently inhibited anti-CD3-induced T-cell proliferation (Figure 2a). IFN-γR KO MSCs had a significantly lower inhibitory capacity (Figure 2a). Proliferation was also measured by analysis of CFSE-labeled CD4+ T cells. Similarly, a lower suppressive capacity of IFN-γR KO MSCs as compared with wild-type MSCs was seen (Figure 2b).


Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis.

Schurgers E, Kelchtermans H, Mitera T, Geboes L, Matthys P - Arthritis Res. Ther. (2010)

Mesenchymal stem cells (MSCs) inhibit the anti-CD3-induced proliferation of CD4+ T cells in vitro. (a) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or interferon-gamma receptor knockout (IFN-γR KO) MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of four cultures ± standard error of the mean (SEM). Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test). (b) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or IFN-γR KO MSCs for 72 hours. The proliferation of CD4+ T cells was analyzed by detection of CFSE dilution by flow cytometry. The percentage inhibition (100 × [(percentage of proliferating CD4+ cells not treated with MSCs -- percentage of proliferating CD4+ cells treated with MSCs)/percentage of proliferating CD4+ cells not treated with MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of three cultures ± SEM. Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test).
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Figure 2: Mesenchymal stem cells (MSCs) inhibit the anti-CD3-induced proliferation of CD4+ T cells in vitro. (a) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or interferon-gamma receptor knockout (IFN-γR KO) MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of four cultures ± standard error of the mean (SEM). Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test). (b) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or IFN-γR KO MSCs for 72 hours. The proliferation of CD4+ T cells was analyzed by detection of CFSE dilution by flow cytometry. The percentage inhibition (100 × [(percentage of proliferating CD4+ cells not treated with MSCs -- percentage of proliferating CD4+ cells treated with MSCs)/percentage of proliferating CD4+ cells not treated with MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of three cultures ± SEM. Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test).
Mentions: To investigate the immunosuppressive potential of MSCs in vitro, we tested their effect on the anti-CD3-induced proliferation of CD4+ T cells. T cells were stimulated in vitro with anti-CD3 antibody in the absence or presence of MSCs and their proliferation was analyzed by thymidine incorporation. MSCs of wild-type origin dose-dependently inhibited anti-CD3-induced T-cell proliferation (Figure 2a). IFN-γR KO MSCs had a significantly lower inhibitory capacity (Figure 2a). Proliferation was also measured by analysis of CFSE-labeled CD4+ T cells. Similarly, a lower suppressive capacity of IFN-γR KO MSCs as compared with wild-type MSCs was seen (Figure 2b).

Bottom Line: In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA.Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, Faculty of Medicine, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. evelien.schurgers@rega.kuleuven.be

ABSTRACT

Introduction: The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-gamma (IFN-gamma) in these processes.

Methods: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-gammaR KO MSC.

Results: Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-gammaR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.

Conclusions: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

Show MeSH
Related in: MedlinePlus