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Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity.

Chatterjee D, Marquardt N, Tufa DM, Beauclair G, Low HZ, Hatlapatka T, Hass R, Kasper C, von Kaisenberg C, Schmidt RE, Jacobs R - Cell Commun. Signal (2014)

Bottom Line: The main soluble immunosuppressant was identified as prostaglandin (PG)-E2.IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.

Results: UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.

Conclusion: Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

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Related in: MedlinePlus

Suppression of NK cell cytotoxicity by UC-MSCs. A: NK cells were cultured alone or with MSCs at NK cell: MSC ratio of 5:1, 10:1, and 20:1. Following overnight co-culture, CD107a degranulation assay was performed with K562 target cells. The bar graphs represent the percentage of CD107a+ NK cells (n = 5) B: NK cells were cultured without MSCs or with MSCs at NK cell: MSC ratio of 10:1. CD107a degranulation assay was performed with K562 target cells (n = 5). Following overnight co-culture, CD107a expression on CD56 bright NK cells was analysed. The bar graphs represent the percentage of CD107a+ cells (n = 9). C, D: NK cells were cultured overnight with or without MSCs. NKG2D expression on the NK cells was analysed by flow cytometry. The bar graphs depict the mean fluorescence intensity (MFI) of NKG2D staining on CD56 dim (C; n = 9) and CD56 bright (D; n = 8) NK cells. E, F, G, H: NK cells were cultured overnight with or without MSCs. Perforin (E; n = 7), granzyme A (F; n = 7), granzyme B (G; n = 5), and granzyme K (H; n = 7) content of the NK cells was analysed by flow cytometry.
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Fig2: Suppression of NK cell cytotoxicity by UC-MSCs. A: NK cells were cultured alone or with MSCs at NK cell: MSC ratio of 5:1, 10:1, and 20:1. Following overnight co-culture, CD107a degranulation assay was performed with K562 target cells. The bar graphs represent the percentage of CD107a+ NK cells (n = 5) B: NK cells were cultured without MSCs or with MSCs at NK cell: MSC ratio of 10:1. CD107a degranulation assay was performed with K562 target cells (n = 5). Following overnight co-culture, CD107a expression on CD56 bright NK cells was analysed. The bar graphs represent the percentage of CD107a+ cells (n = 9). C, D: NK cells were cultured overnight with or without MSCs. NKG2D expression on the NK cells was analysed by flow cytometry. The bar graphs depict the mean fluorescence intensity (MFI) of NKG2D staining on CD56 dim (C; n = 9) and CD56 bright (D; n = 8) NK cells. E, F, G, H: NK cells were cultured overnight with or without MSCs. Perforin (E; n = 7), granzyme A (F; n = 7), granzyme B (G; n = 5), and granzyme K (H; n = 7) content of the NK cells was analysed by flow cytometry.

Mentions: To examine the effect of MSCs on NK cells, freshly isolated, unstimulated NK cells were incubated with MSCs in direct contact at ratio 5:1, 10:1, and 20:1 for 16 hours. Following the co-culture, NK cells were harvested and exposed to K652 target cells to trigger NK cell degranulation. Surface expression of CD107a was analysed as a marker for degranulation. We found that NK cells pre-cultured with MSCs had significantly reduced degranulation capacity compared to NK cells cultured alone (Figure 2A). There was no significant difference in the suppression induced by different ratios of MSC during co-culture (Figure 2A). For all subsequent experiments, the NK: MSC ratio of 10: 1 was used. To confirm that the freshly isolated NK cells did not kill the MSCs during the co-culture, we performed chromium release assay with MSCs as target cells. We observed that the NK cells could efficiently lyse K562 cells but not the MSCs (Additional file 1: Figure S1). IL-15 pre-activated NK cells were better at killing the MSCs but the lysis was still less when compared to K562 (Additional file 1: Figure S1).Figure 2


Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity.

Chatterjee D, Marquardt N, Tufa DM, Beauclair G, Low HZ, Hatlapatka T, Hass R, Kasper C, von Kaisenberg C, Schmidt RE, Jacobs R - Cell Commun. Signal (2014)

Suppression of NK cell cytotoxicity by UC-MSCs. A: NK cells were cultured alone or with MSCs at NK cell: MSC ratio of 5:1, 10:1, and 20:1. Following overnight co-culture, CD107a degranulation assay was performed with K562 target cells. The bar graphs represent the percentage of CD107a+ NK cells (n = 5) B: NK cells were cultured without MSCs or with MSCs at NK cell: MSC ratio of 10:1. CD107a degranulation assay was performed with K562 target cells (n = 5). Following overnight co-culture, CD107a expression on CD56 bright NK cells was analysed. The bar graphs represent the percentage of CD107a+ cells (n = 9). C, D: NK cells were cultured overnight with or without MSCs. NKG2D expression on the NK cells was analysed by flow cytometry. The bar graphs depict the mean fluorescence intensity (MFI) of NKG2D staining on CD56 dim (C; n = 9) and CD56 bright (D; n = 8) NK cells. E, F, G, H: NK cells were cultured overnight with or without MSCs. Perforin (E; n = 7), granzyme A (F; n = 7), granzyme B (G; n = 5), and granzyme K (H; n = 7) content of the NK cells was analysed by flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4195898&req=5

Fig2: Suppression of NK cell cytotoxicity by UC-MSCs. A: NK cells were cultured alone or with MSCs at NK cell: MSC ratio of 5:1, 10:1, and 20:1. Following overnight co-culture, CD107a degranulation assay was performed with K562 target cells. The bar graphs represent the percentage of CD107a+ NK cells (n = 5) B: NK cells were cultured without MSCs or with MSCs at NK cell: MSC ratio of 10:1. CD107a degranulation assay was performed with K562 target cells (n = 5). Following overnight co-culture, CD107a expression on CD56 bright NK cells was analysed. The bar graphs represent the percentage of CD107a+ cells (n = 9). C, D: NK cells were cultured overnight with or without MSCs. NKG2D expression on the NK cells was analysed by flow cytometry. The bar graphs depict the mean fluorescence intensity (MFI) of NKG2D staining on CD56 dim (C; n = 9) and CD56 bright (D; n = 8) NK cells. E, F, G, H: NK cells were cultured overnight with or without MSCs. Perforin (E; n = 7), granzyme A (F; n = 7), granzyme B (G; n = 5), and granzyme K (H; n = 7) content of the NK cells was analysed by flow cytometry.
Mentions: To examine the effect of MSCs on NK cells, freshly isolated, unstimulated NK cells were incubated with MSCs in direct contact at ratio 5:1, 10:1, and 20:1 for 16 hours. Following the co-culture, NK cells were harvested and exposed to K652 target cells to trigger NK cell degranulation. Surface expression of CD107a was analysed as a marker for degranulation. We found that NK cells pre-cultured with MSCs had significantly reduced degranulation capacity compared to NK cells cultured alone (Figure 2A). There was no significant difference in the suppression induced by different ratios of MSC during co-culture (Figure 2A). For all subsequent experiments, the NK: MSC ratio of 10: 1 was used. To confirm that the freshly isolated NK cells did not kill the MSCs during the co-culture, we performed chromium release assay with MSCs as target cells. We observed that the NK cells could efficiently lyse K562 cells but not the MSCs (Additional file 1: Figure S1). IL-15 pre-activated NK cells were better at killing the MSCs but the lysis was still less when compared to K562 (Additional file 1: Figure S1).Figure 2

Bottom Line: The main soluble immunosuppressant was identified as prostaglandin (PG)-E2.IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.

Results: UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.

Conclusion: Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

Show MeSH
Related in: MedlinePlus