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Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

Bruno S, Grange C, Tapparo M, Pasquino C, Romagnoli R, Dametto E, Amoroso A, Tetta C, Camussi G - Stem Cells Int (2016)

Bottom Line: At variance with MSCs, HLSCs did not elicit NK degranulation.Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release.This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology and Health Science, University of Torino, 10126 Torino, Italy.

ABSTRACT
Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

No MeSH data available.


Related in: MedlinePlus

HLSCs suppress T-lymphocyte proliferation through IDO and PGE2 activity. (a) CD3+ lymphocytes were stimulated with PMA, in the presence or the absence of MSCs or HLSCs at different ratio (lymphocytes: HLSCs/MSCs 1 : 1, 2 : 1, 5 : 1, and 10 : 1) in direct contact or in the presence of transwells. The proliferation rate was evaluated after 3 days of coculture. Results are expressed as mean ± SD of 6 different experiments conducted in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by nonparametric Wilcoxon test: ∗p < 0.05 lymphocytes stimulated with PMA cocultured in contact with HLSCs versus lymphocytes stimulated with PMA cocultured in contact with MSCs; #p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs, in the presence of transwells, versus lymphocytes stimulated with PMA cocultured with MSCs, in the presence of transwells. (b) IDO and PGE2 are involved in HLSC suppression of T-cell proliferation. CD3+ cells were stimulated with PMA in the presence of HLSCs (5 : 1 ratio) with or without inhibitors of IDO (1-methyl-L-tryptophan) and Cox-1 and/or Cox-2 (indomethacin, NS-398) in the presence of transwells. The proliferation rate was evaluated after 3 days. Results are expressed as mean ± SD of 6 different experiments in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by ANOVA with Bonferroni correction: ∗p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs and different inhibitors versus lymphocytes stimulated with PMA cocultured with HLSCs. (c) and (d) mRNA expression levels of Cox-2 were evaluated in HLSCs (c) and MSCs (d) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. (e) and (f) mRNA expression levels of IDO were evaluated in HLSCs (e) and MSCs (f) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. Cox-2 and IDO expression levels increased after coculture with activated lymphocytes and after stimulation with CM. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by ANOVA with Bonferroni correction, ∗p < 0.05 gene expression of MSCs or HLSCs after coculture with CD3+ cells stimulated with PMA or CD3/CD28 antibodies or after culture with CM versus MSCs or HLSCs alone. (g) mRNA expression levels of COX-1 were evaluated in MSCs and HLSCs. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by Student's t-test (unpaired, 2-tailed); ∗p < 0.05 COX-1 expression of HLSCs versus MSCs. (h) Cell supernatants from cocultures were harvested to detect PGE2 production by ELISA. A significant increased production of PGE2 (p < 0.05) was observed after 3 days of coculture of CD3+ cells stimulated with PMA and cocultured with HLSCs.
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fig1: HLSCs suppress T-lymphocyte proliferation through IDO and PGE2 activity. (a) CD3+ lymphocytes were stimulated with PMA, in the presence or the absence of MSCs or HLSCs at different ratio (lymphocytes: HLSCs/MSCs 1 : 1, 2 : 1, 5 : 1, and 10 : 1) in direct contact or in the presence of transwells. The proliferation rate was evaluated after 3 days of coculture. Results are expressed as mean ± SD of 6 different experiments conducted in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by nonparametric Wilcoxon test: ∗p < 0.05 lymphocytes stimulated with PMA cocultured in contact with HLSCs versus lymphocytes stimulated with PMA cocultured in contact with MSCs; #p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs, in the presence of transwells, versus lymphocytes stimulated with PMA cocultured with MSCs, in the presence of transwells. (b) IDO and PGE2 are involved in HLSC suppression of T-cell proliferation. CD3+ cells were stimulated with PMA in the presence of HLSCs (5 : 1 ratio) with or without inhibitors of IDO (1-methyl-L-tryptophan) and Cox-1 and/or Cox-2 (indomethacin, NS-398) in the presence of transwells. The proliferation rate was evaluated after 3 days. Results are expressed as mean ± SD of 6 different experiments in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by ANOVA with Bonferroni correction: ∗p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs and different inhibitors versus lymphocytes stimulated with PMA cocultured with HLSCs. (c) and (d) mRNA expression levels of Cox-2 were evaluated in HLSCs (c) and MSCs (d) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. (e) and (f) mRNA expression levels of IDO were evaluated in HLSCs (e) and MSCs (f) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. Cox-2 and IDO expression levels increased after coculture with activated lymphocytes and after stimulation with CM. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by ANOVA with Bonferroni correction, ∗p < 0.05 gene expression of MSCs or HLSCs after coculture with CD3+ cells stimulated with PMA or CD3/CD28 antibodies or after culture with CM versus MSCs or HLSCs alone. (g) mRNA expression levels of COX-1 were evaluated in MSCs and HLSCs. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by Student's t-test (unpaired, 2-tailed); ∗p < 0.05 COX-1 expression of HLSCs versus MSCs. (h) Cell supernatants from cocultures were harvested to detect PGE2 production by ELISA. A significant increased production of PGE2 (p < 0.05) was observed after 3 days of coculture of CD3+ cells stimulated with PMA and cocultured with HLSCs.

Mentions: To determine the capacity of HLSCs to interfere with T-lymphocyte proliferation, HLSCs were cocultured with allogeneic CD3+ cells activated or not with PMA. No proliferation was noted when purified T-lymphocytes were cocultured in the presence of HLSCs or MSCs, at different ratios (Supplementary Figure 1). Indeed, after 3 days of coculture, HLSCs inhibited PMA-induced CD3+ cell proliferation in a dose-dependent manner. In cell contact conditions, at a 5 : 1 lymphocyte : stem cell ratio, and in the presence of transwells, the inhibitory effect of HLSCs was significantly greater than that of MSCs (Figure 1(a) and Supplementary Figure 1). Cell contact was not required for HLSC suppression of T-cell proliferation, as HLSCs separated by a transwell were almost as effective as those in direct contact with lymphocytes (Figure 1(a) and Supplementary Figure 1). To confirm the capacity of HLSCs to interfere with T-cell proliferation, we also stimulated transwell cultures with anti-CD3 and CD28 antibodies and observed an inhibition of T-cell proliferation in the presence of HLSCs at different cell ratios (Supplementary Figure 2).


Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

Bruno S, Grange C, Tapparo M, Pasquino C, Romagnoli R, Dametto E, Amoroso A, Tetta C, Camussi G - Stem Cells Int (2016)

HLSCs suppress T-lymphocyte proliferation through IDO and PGE2 activity. (a) CD3+ lymphocytes were stimulated with PMA, in the presence or the absence of MSCs or HLSCs at different ratio (lymphocytes: HLSCs/MSCs 1 : 1, 2 : 1, 5 : 1, and 10 : 1) in direct contact or in the presence of transwells. The proliferation rate was evaluated after 3 days of coculture. Results are expressed as mean ± SD of 6 different experiments conducted in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by nonparametric Wilcoxon test: ∗p < 0.05 lymphocytes stimulated with PMA cocultured in contact with HLSCs versus lymphocytes stimulated with PMA cocultured in contact with MSCs; #p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs, in the presence of transwells, versus lymphocytes stimulated with PMA cocultured with MSCs, in the presence of transwells. (b) IDO and PGE2 are involved in HLSC suppression of T-cell proliferation. CD3+ cells were stimulated with PMA in the presence of HLSCs (5 : 1 ratio) with or without inhibitors of IDO (1-methyl-L-tryptophan) and Cox-1 and/or Cox-2 (indomethacin, NS-398) in the presence of transwells. The proliferation rate was evaluated after 3 days. Results are expressed as mean ± SD of 6 different experiments in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by ANOVA with Bonferroni correction: ∗p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs and different inhibitors versus lymphocytes stimulated with PMA cocultured with HLSCs. (c) and (d) mRNA expression levels of Cox-2 were evaluated in HLSCs (c) and MSCs (d) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. (e) and (f) mRNA expression levels of IDO were evaluated in HLSCs (e) and MSCs (f) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. Cox-2 and IDO expression levels increased after coculture with activated lymphocytes and after stimulation with CM. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by ANOVA with Bonferroni correction, ∗p < 0.05 gene expression of MSCs or HLSCs after coculture with CD3+ cells stimulated with PMA or CD3/CD28 antibodies or after culture with CM versus MSCs or HLSCs alone. (g) mRNA expression levels of COX-1 were evaluated in MSCs and HLSCs. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by Student's t-test (unpaired, 2-tailed); ∗p < 0.05 COX-1 expression of HLSCs versus MSCs. (h) Cell supernatants from cocultures were harvested to detect PGE2 production by ELISA. A significant increased production of PGE2 (p < 0.05) was observed after 3 days of coculture of CD3+ cells stimulated with PMA and cocultured with HLSCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4834412&req=5

fig1: HLSCs suppress T-lymphocyte proliferation through IDO and PGE2 activity. (a) CD3+ lymphocytes were stimulated with PMA, in the presence or the absence of MSCs or HLSCs at different ratio (lymphocytes: HLSCs/MSCs 1 : 1, 2 : 1, 5 : 1, and 10 : 1) in direct contact or in the presence of transwells. The proliferation rate was evaluated after 3 days of coculture. Results are expressed as mean ± SD of 6 different experiments conducted in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by nonparametric Wilcoxon test: ∗p < 0.05 lymphocytes stimulated with PMA cocultured in contact with HLSCs versus lymphocytes stimulated with PMA cocultured in contact with MSCs; #p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs, in the presence of transwells, versus lymphocytes stimulated with PMA cocultured with MSCs, in the presence of transwells. (b) IDO and PGE2 are involved in HLSC suppression of T-cell proliferation. CD3+ cells were stimulated with PMA in the presence of HLSCs (5 : 1 ratio) with or without inhibitors of IDO (1-methyl-L-tryptophan) and Cox-1 and/or Cox-2 (indomethacin, NS-398) in the presence of transwells. The proliferation rate was evaluated after 3 days. Results are expressed as mean ± SD of 6 different experiments in duplicate. 100% of proliferation corresponds to lymphocytes stimulated with PMA. Data were analysed by ANOVA with Bonferroni correction: ∗p < 0.05 lymphocytes stimulated with PMA cocultured with HLSCs and different inhibitors versus lymphocytes stimulated with PMA cocultured with HLSCs. (c) and (d) mRNA expression levels of Cox-2 were evaluated in HLSCs (c) and MSCs (d) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. (e) and (f) mRNA expression levels of IDO were evaluated in HLSCs (e) and MSCs (f) unstimulated, stimulated with PMA, after 3 days of coculture with CD3+ lymphocytes stimulated with PMA or with CD3/CD28 antibodies, stimulated with CM from T-cells stimulated with PMA, and cultured with unstimulated lymphocytes. Cox-2 and IDO expression levels increased after coculture with activated lymphocytes and after stimulation with CM. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by ANOVA with Bonferroni correction, ∗p < 0.05 gene expression of MSCs or HLSCs after coculture with CD3+ cells stimulated with PMA or CD3/CD28 antibodies or after culture with CM versus MSCs or HLSCs alone. (g) mRNA expression levels of COX-1 were evaluated in MSCs and HLSCs. Data are expressed as relative quantification using ΔΔCt method. Normalization was made using actin as housekeeping gene. Data were analysed by Student's t-test (unpaired, 2-tailed); ∗p < 0.05 COX-1 expression of HLSCs versus MSCs. (h) Cell supernatants from cocultures were harvested to detect PGE2 production by ELISA. A significant increased production of PGE2 (p < 0.05) was observed after 3 days of coculture of CD3+ cells stimulated with PMA and cocultured with HLSCs.
Mentions: To determine the capacity of HLSCs to interfere with T-lymphocyte proliferation, HLSCs were cocultured with allogeneic CD3+ cells activated or not with PMA. No proliferation was noted when purified T-lymphocytes were cocultured in the presence of HLSCs or MSCs, at different ratios (Supplementary Figure 1). Indeed, after 3 days of coculture, HLSCs inhibited PMA-induced CD3+ cell proliferation in a dose-dependent manner. In cell contact conditions, at a 5 : 1 lymphocyte : stem cell ratio, and in the presence of transwells, the inhibitory effect of HLSCs was significantly greater than that of MSCs (Figure 1(a) and Supplementary Figure 1). Cell contact was not required for HLSC suppression of T-cell proliferation, as HLSCs separated by a transwell were almost as effective as those in direct contact with lymphocytes (Figure 1(a) and Supplementary Figure 1). To confirm the capacity of HLSCs to interfere with T-cell proliferation, we also stimulated transwell cultures with anti-CD3 and CD28 antibodies and observed an inhibition of T-cell proliferation in the presence of HLSCs at different cell ratios (Supplementary Figure 2).

Bottom Line: At variance with MSCs, HLSCs did not elicit NK degranulation.Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release.This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology and Health Science, University of Torino, 10126 Torino, Italy.

ABSTRACT
Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

No MeSH data available.


Related in: MedlinePlus