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Background: Mesenchymal stem cells (MSCs) display active capacities of suppressing or modulating harmful immune responses through diverse molecular mechanisms. These cells are under extensive translational efforts as cell therapies for immune-mediated diseases and transplantations. A wide range of preclinical studies and limited number of clinical trials using MSCs have not only shown promising safety and efficacy profiles but have also revealed changes in regulatory T cell (T reg) frequency and function. However, the mechanisms underlying this important observation are not well understood. Cell-to-cell contact, production of soluble factors, reprogramming of antigen presenting cells to tolerogenic phenotypes have emerged as possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion. We and others demonstrated that adult bone-marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4+ (“helper”) and CD8+ (“cytotoxic”) T cells but also indirectly through induction of Tregs. In parallel we demonstrated that fetal liver (FL)-MSCs displays much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4+ and CD8+ T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs.
Methods: MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation and their proliferation potential. Using different in-vitro combinations, we performed co-cultures of FL or BM-MSCs and murine CD3+CD25-T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs.
Results: We demonstrated that although both types of MSC exhibit similar phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4+ and CD8+ murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4+CD25+Foxp3+ T regs compared to BM-MSCs.
Conclusions: These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction.
Keywords
Fetal Liver, Adult Bone Marrow, Mesenchymal Stem Cells, T cell Immunomodulation, Induced regulatory T cells
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.tiff
Supplementary Figure 1: FL-MSCs contained both CD271bright and CD271low cells, while adult BM –MSCs contained only CD271low cells FL and BM MSCs cultured at passage 4, were stained with APC-anti-CD271 Mab. Representative flow cytometry histogram and dot plots show the relative expression of CD271 by FL and BM-MSCs. Numbers indicate the percentage of CD271bright cells in the corresponding histogram bars and quadrants. Data are representative of 3 independent experiments (n=3).
- SupplementaryFigure2.tiff
Supplementary Figure 2: In-vitro osteogenic and adipogenic differentiation capacity of FL-MSCs compared to BM-MSCs FL (passage 4) and BM (passage4)-MSCs were cultured with or without inductive media to induce osteogenic or adipogenic cell differentiation. Representative images of osteogenic and adipogenic differentiation detected by Alizarin Red S and Oil Red O staining, respectively. Data are representative of 2 independent experiments (n=6). Scale bar indicates 100 μm.
- SupplementaryFigure3.tiff
Supplementary Figure 3: Long-lasting down-modulation of CD4+ and CD8+ T convs by FL-MSCs compare to BM-MSCs CD3/CD28 activated CD3+CD25- effector T cells were co-cultured with BM-MSCs or FL-MSCs in a fixed 1:5 MSC to T cell ratio. After 3day, T cells were collected and T cell activation markers (CD25, GITR, ICOS and TNFR2) were analyzed by flow cytometry. Statistical summary dot-plot graphs showing the percentage each marker analyzed in CD4+Foxp3- (A) or CD8+Foxp3- (B) T convs. Each dot represents a measured value collected from 2 different experiments (n=12 for T cells + Beads group (black) and n=9 for BM-MSCs + T cells (red) and FL-MSCs + T cells (blue) groups). For each group of values, horizontal lines represent mean value ± SEM. One way ANOVA analysis was performed to generate P values. ns: non-significant, *P<.05, **P<.01, ***P<.001, ****P<.0001. Beads: Anti-CD3 and anti-CD28 activation Beads; T convs: Conventional T cells.
- SupplementaryFigure4.tiff
Supplemental Figure 4: FL-MSCs are more immunosuppressive against human HLA mismatched T cells than BM-MSCs. CFSE labelled, CD3/CD28 activated CD3+CD25- human effector T cells were co-cultured with FL-MSCs or BM-MSCs in 6 different MSC to T cell ratios. After 3 days, proliferation of CD4+ (A) and CD8+ T cells (B) was measured by flow cytometry based on CFSE dilution. Each bar represents the percent of dividing cells. The first bar represents the unstimulated T cells alone (n=4), the second bar represents the CD3/CD28-stimulated T cells alone (n=4). Further bars depict T cells co-cultured with either BM-MSCs in red (n=4) or FL-MSCs in blue (n=4). Data are represented as mean value ± SEM. One way ANOVA analysis was performed to generate P values. ns: non-significant, *P<.05, **P<.01, ***P<.001, ****P<.0001. Beads: Anti-CD3 and anti-CD28 activation Beads; TCs: T cells; T cons: Conventional T cells.
- SupplementaryTable1.docx
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CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 2
Posted 18 Nov, 2020
No community comments so far
Editorial decision: Accept
On 15 Dec, 2020
Review #2 received
Received 12 Dec, 2020
Reviewer #2 agreed
On 21 Nov, 2020
Review #1 received
Received 18 Nov, 2020
Reviewer #1 agreed
On 16 Nov, 2020
Reviewers invited
Invitations sent on 15 Nov, 2020
Editor assigned
On 12 Nov, 2020
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On 12 Nov, 2020
Editor invited
On 12 Nov, 2020
No comments provided
Editorial decision: Minor Revision
On 18 Oct, 2020
Review #2 received
Received 15 Oct, 2020
Reviewer #2 agreed
On 29 Sep, 2020
Review #1 received
Received 22 Aug, 2020
Reviewer #1 agreed
On 17 Aug, 2020
Reviewers invited
Invitations sent on 14 Aug, 2020
Editor assigned
On 12 Jul, 2020
Editor invited
On 11 Jul, 2020
Submission checks complete
On 07 Jul, 2020
First submitted
On 03 Jul, 2020
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Subject Areas
Stem Cell & Developmental Cell Biology
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A new preprint design is coming!
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See the published version of this preprint at Stem Cell Research & Therapy.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
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Peer Review Timeline
CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 2
Posted 18 Nov, 2020
No community comments so far
Editorial decision: Accept
On 15 Dec, 2020
Review #2 received
Received 12 Dec, 2020
Reviewer #2 agreed
On 21 Nov, 2020
Review #1 received
Received 18 Nov, 2020
Reviewer #1 agreed
On 16 Nov, 2020
Reviewers invited
Invitations sent on 15 Nov, 2020
Editor assigned
On 12 Nov, 2020
Submission checks complete
On 12 Nov, 2020
Editor invited
On 12 Nov, 2020
No comments provided
Editorial decision: Minor Revision
On 18 Oct, 2020
Review #2 received
Received 15 Oct, 2020
Reviewer #2 agreed
On 29 Sep, 2020
Review #1 received
Received 22 Aug, 2020
Reviewer #1 agreed
On 17 Aug, 2020
Reviewers invited
Invitations sent on 14 Aug, 2020
Editor assigned
On 12 Jul, 2020
Editor invited
On 11 Jul, 2020
Submission checks complete
On 07 Jul, 2020
First submitted
On 03 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Stem Cell & Developmental Cell Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Stem Cell Research & Therapy.
Background: Mesenchymal stem cells (MSCs) display active capacities of suppressing or modulating harmful immune responses through diverse molecular mechanisms. These cells are under extensive translational efforts as cell therapies for immune-mediated diseases and transplantations. A wide range of preclinical studies and limited number of clinical trials using MSCs have not only shown promising safety and efficacy profiles but have also revealed changes in regulatory T cell (T reg) frequency and function. However, the mechanisms underlying this important observation are not well understood. Cell-to-cell contact, production of soluble factors, reprogramming of antigen presenting cells to tolerogenic phenotypes have emerged as possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion. We and others demonstrated that adult bone-marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4+ (“helper”) and CD8+ (“cytotoxic”) T cells but also indirectly through induction of Tregs. In parallel we demonstrated that fetal liver (FL)-MSCs displays much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4+ and CD8+ T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs.
Methods: MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation and their proliferation potential. Using different in-vitro combinations, we performed co-cultures of FL or BM-MSCs and murine CD3+CD25-T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs.
Results: We demonstrated that although both types of MSC exhibit similar phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4+ and CD8+ murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4+CD25+Foxp3+ T regs compared to BM-MSCs.
Conclusions: These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigure1.tiff
Supplementary Figure 1: FL-MSCs contained both CD271bright and CD271low cells, while adult BM –MSCs contained only CD271low cells FL and BM MSCs cultured at passage 4, were stained with APC-anti-CD271 Mab. Representative flow cytometry histogram and dot plots show the relative expression of CD271 by FL and BM-MSCs. Numbers indicate the percentage of CD271bright cells in the corresponding histogram bars and quadrants. Data are representative of 3 independent experiments (n=3).
- SupplementaryFigure2.tiff
Supplementary Figure 2: In-vitro osteogenic and adipogenic differentiation capacity of FL-MSCs compared to BM-MSCs FL (passage 4) and BM (passage4)-MSCs were cultured with or without inductive media to induce osteogenic or adipogenic cell differentiation. Representative images of osteogenic and adipogenic differentiation detected by Alizarin Red S and Oil Red O staining, respectively. Data are representative of 2 independent experiments (n=6). Scale bar indicates 100 μm.
- SupplementaryFigure3.tiff
Supplementary Figure 3: Long-lasting down-modulation of CD4+ and CD8+ T convs by FL-MSCs compare to BM-MSCs CD3/CD28 activated CD3+CD25- effector T cells were co-cultured with BM-MSCs or FL-MSCs in a fixed 1:5 MSC to T cell ratio. After 3day, T cells were collected and T cell activation markers (CD25, GITR, ICOS and TNFR2) were analyzed by flow cytometry. Statistical summary dot-plot graphs showing the percentage each marker analyzed in CD4+Foxp3- (A) or CD8+Foxp3- (B) T convs. Each dot represents a measured value collected from 2 different experiments (n=12 for T cells + Beads group (black) and n=9 for BM-MSCs + T cells (red) and FL-MSCs + T cells (blue) groups). For each group of values, horizontal lines represent mean value ± SEM. One way ANOVA analysis was performed to generate P values. ns: non-significant, *P<.05, **P<.01, ***P<.001, ****P<.0001. Beads: Anti-CD3 and anti-CD28 activation Beads; T convs: Conventional T cells.
- SupplementaryFigure4.tiff
Supplemental Figure 4: FL-MSCs are more immunosuppressive against human HLA mismatched T cells than BM-MSCs. CFSE labelled, CD3/CD28 activated CD3+CD25- human effector T cells were co-cultured with FL-MSCs or BM-MSCs in 6 different MSC to T cell ratios. After 3 days, proliferation of CD4+ (A) and CD8+ T cells (B) was measured by flow cytometry based on CFSE dilution. Each bar represents the percent of dividing cells. The first bar represents the unstimulated T cells alone (n=4), the second bar represents the CD3/CD28-stimulated T cells alone (n=4). Further bars depict T cells co-cultured with either BM-MSCs in red (n=4) or FL-MSCs in blue (n=4). Data are represented as mean value ± SEM. One way ANOVA analysis was performed to generate P values. ns: non-significant, *P<.05, **P<.01, ***P<.001, ****P<.0001. Beads: Anti-CD3 and anti-CD28 activation Beads; TCs: T cells; T cons: Conventional T cells.
- SupplementaryTable1.docx
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