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Induction of macrophage-like immunosuppressive cells from mouse ES cells that contribute to prolong allogeneic graft survival.

Kudo H, Wada H, Sasaki H, Tsuji H, Otsuka R, Baghdadi M, Kojo S, Chikaraishi T, Seino K - PLoS ONE (2014)

Bottom Line: Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response.Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule.Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; Department of Urology St. Marianna University School of Medicine, Miyamae-ku, Kawasaki City, Kanagawa, Japan.

ABSTRACT
Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.

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ES-SCs suppress T cell proliferation.(A) MLR assay: C3H T cells vs. bone marrow-derived DCs. Where indicated, 129 ES-DCs or ES-SCs were added to MLR culture and inhibitory effects were measured by proliferation rate (CFSE cell division). (B) ES-SCs and MLR assay: C3H T cells were stimulated with irradiated bone marrow-derived DCs, and then graded numbers of ES-SCs were added to MLR culture. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (C) ES-SCs and MLR assay. Several immunosuppressive molecules were blocked by specific inhibitors: Anti-TGFβ mAb (10 µg/mL), anti-PD-L2 mAb (10 µg/mL), L-NMMA (50 µM), or corresponding isotype-matched controls were added. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (D) Measurement of NO concentration in the supernatants of ES-DCs or ES-SCs. (E) Specificity of immune suppression by ES-SCs. Splenic T cells from ES-SCs- or PBS-administered C3H mice were stimulated with bone marrow-derived DCs from the indicated mice including C3H (Syn; Syngeneic), 129 (Allo; allogeneic but same background as ES-SCs), and BALB/c (3rd; allogeneic, third party). T cell proliferation was estimated by [3H] thymidine uptake. Data represent mean ± SD of three wells. Similar results were obtained from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.
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pone-0111826-g002: ES-SCs suppress T cell proliferation.(A) MLR assay: C3H T cells vs. bone marrow-derived DCs. Where indicated, 129 ES-DCs or ES-SCs were added to MLR culture and inhibitory effects were measured by proliferation rate (CFSE cell division). (B) ES-SCs and MLR assay: C3H T cells were stimulated with irradiated bone marrow-derived DCs, and then graded numbers of ES-SCs were added to MLR culture. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (C) ES-SCs and MLR assay. Several immunosuppressive molecules were blocked by specific inhibitors: Anti-TGFβ mAb (10 µg/mL), anti-PD-L2 mAb (10 µg/mL), L-NMMA (50 µM), or corresponding isotype-matched controls were added. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (D) Measurement of NO concentration in the supernatants of ES-DCs or ES-SCs. (E) Specificity of immune suppression by ES-SCs. Splenic T cells from ES-SCs- or PBS-administered C3H mice were stimulated with bone marrow-derived DCs from the indicated mice including C3H (Syn; Syngeneic), 129 (Allo; allogeneic but same background as ES-SCs), and BALB/c (3rd; allogeneic, third party). T cell proliferation was estimated by [3H] thymidine uptake. Data represent mean ± SD of three wells. Similar results were obtained from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Mentions: As we anticipated obtaining immune-regulatory cells from PSCs, we next examined whether either ES-DCs or ES-SCs could interfere with allogeneic immune response. To do so, we stimulated T cells isolated from C3H/HeSlc (C3H; H-2k) mice with allogeneic bone marrow-derived DCs of 129 mice (H-2b) (mixed lymphocyte reaction; MLR) in the presence or absence of either ES-DCs or ES-SCs. We found that the addition of ES-SCs significantly inhibited T cell response, whereas ES-DCs were almost ineffective (Fig. 2A). To examine antigen-presenting capacity of the cells, ES-SCs or ES-DCs were used as stimulators of MLR instead of bone marrow-derived DCs. Interestingly; a significant proliferation of C3H T cells was observed when co-cultured with ES-DCs but not ES-SCs (Fig. 2A). Importantly, ES-SCs could remarkably inhibit T cell proliferation at a low ratio of 1/10: ES-SCs/T cell (Fig. 2B). These results indicate that ES-SCs, but not ES-DCs, possess a capacity to inhibit allogeneic T cell responses.


Induction of macrophage-like immunosuppressive cells from mouse ES cells that contribute to prolong allogeneic graft survival.

Kudo H, Wada H, Sasaki H, Tsuji H, Otsuka R, Baghdadi M, Kojo S, Chikaraishi T, Seino K - PLoS ONE (2014)

ES-SCs suppress T cell proliferation.(A) MLR assay: C3H T cells vs. bone marrow-derived DCs. Where indicated, 129 ES-DCs or ES-SCs were added to MLR culture and inhibitory effects were measured by proliferation rate (CFSE cell division). (B) ES-SCs and MLR assay: C3H T cells were stimulated with irradiated bone marrow-derived DCs, and then graded numbers of ES-SCs were added to MLR culture. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (C) ES-SCs and MLR assay. Several immunosuppressive molecules were blocked by specific inhibitors: Anti-TGFβ mAb (10 µg/mL), anti-PD-L2 mAb (10 µg/mL), L-NMMA (50 µM), or corresponding isotype-matched controls were added. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (D) Measurement of NO concentration in the supernatants of ES-DCs or ES-SCs. (E) Specificity of immune suppression by ES-SCs. Splenic T cells from ES-SCs- or PBS-administered C3H mice were stimulated with bone marrow-derived DCs from the indicated mice including C3H (Syn; Syngeneic), 129 (Allo; allogeneic but same background as ES-SCs), and BALB/c (3rd; allogeneic, third party). T cell proliferation was estimated by [3H] thymidine uptake. Data represent mean ± SD of three wells. Similar results were obtained from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.
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getmorefigures.php?uid=PMC4214817&req=5

pone-0111826-g002: ES-SCs suppress T cell proliferation.(A) MLR assay: C3H T cells vs. bone marrow-derived DCs. Where indicated, 129 ES-DCs or ES-SCs were added to MLR culture and inhibitory effects were measured by proliferation rate (CFSE cell division). (B) ES-SCs and MLR assay: C3H T cells were stimulated with irradiated bone marrow-derived DCs, and then graded numbers of ES-SCs were added to MLR culture. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (C) ES-SCs and MLR assay. Several immunosuppressive molecules were blocked by specific inhibitors: Anti-TGFβ mAb (10 µg/mL), anti-PD-L2 mAb (10 µg/mL), L-NMMA (50 µM), or corresponding isotype-matched controls were added. T cell proliferation was estimated by [3H] thymidine uptake. Results are expressed as mean cpm ± SD. (D) Measurement of NO concentration in the supernatants of ES-DCs or ES-SCs. (E) Specificity of immune suppression by ES-SCs. Splenic T cells from ES-SCs- or PBS-administered C3H mice were stimulated with bone marrow-derived DCs from the indicated mice including C3H (Syn; Syngeneic), 129 (Allo; allogeneic but same background as ES-SCs), and BALB/c (3rd; allogeneic, third party). T cell proliferation was estimated by [3H] thymidine uptake. Data represent mean ± SD of three wells. Similar results were obtained from three independent experiments. *P<0.05, **P<0.01, ***P<0.001.
Mentions: As we anticipated obtaining immune-regulatory cells from PSCs, we next examined whether either ES-DCs or ES-SCs could interfere with allogeneic immune response. To do so, we stimulated T cells isolated from C3H/HeSlc (C3H; H-2k) mice with allogeneic bone marrow-derived DCs of 129 mice (H-2b) (mixed lymphocyte reaction; MLR) in the presence or absence of either ES-DCs or ES-SCs. We found that the addition of ES-SCs significantly inhibited T cell response, whereas ES-DCs were almost ineffective (Fig. 2A). To examine antigen-presenting capacity of the cells, ES-SCs or ES-DCs were used as stimulators of MLR instead of bone marrow-derived DCs. Interestingly; a significant proliferation of C3H T cells was observed when co-cultured with ES-DCs but not ES-SCs (Fig. 2A). Importantly, ES-SCs could remarkably inhibit T cell proliferation at a low ratio of 1/10: ES-SCs/T cell (Fig. 2B). These results indicate that ES-SCs, but not ES-DCs, possess a capacity to inhibit allogeneic T cell responses.

Bottom Line: Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response.Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule.Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; Department of Urology St. Marianna University School of Medicine, Miyamae-ku, Kawasaki City, Kanagawa, Japan.

ABSTRACT
Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.

Show MeSH
Related in: MedlinePlus