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Keywords:

  • Immune regulation;
  • Mesenchymal stem cells;
  • Notch1;
  • Tolerance;
  • Treg cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Notch1 signaling is involved in regulatory T (Treg)-cell differentiation. We previously demonstrated that, when cocultured with CD3+ cells, mesenchymal stem cells (MSCs) induced a T-cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4+ T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI-I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC-induced CD4+CD25highFOXP3+ cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF-β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg-cell induction mediated by MSCs.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Regulatory T (Treg) cells play an important role in peripheral self-tolerance in humans [1] and Treg-cell-based clinical trials have recently been reported [2]. Treg-cell suppressor populations are considered to be CD4+CD25highFOXP3+ T cells that develop in the thymus (natural Treg (nTreg) cells) or T cells that are induced from peripheral naïve T cells (inducible Treg (iTreg) cells). iTreg cells can be recruited by MSCs from CD4+ T cells either in-directly, by means of tolerogenic DCs [3, 4], or directly by MSC T-cell interactions requiring cell contact [4, 5] and soluble factors such as prostaglandin E2, IL-6, IL-10, indoleamine 2.3 dioxy-genase (IDO), transforming growth factor (TGF-β1) [3, 4, 6], and heme oxygenase-1 [7]. In vivo and in vitro studies indicated that Treg-cell induction from conventional CD4+ T cells is important in MSCs-mediated immunomodulation [4, 8, 9].

Notch family members have been implicated in the differentiation of various CD4+ T-cell subsets, including Th1 [10], Th2 [11], and Treg cells [12, 13] through over-expression of the Notch ligand Jagged1 [13, 14]. Recent studies showed the presence of Notch ligands in MSCs. However, no studies have linked the Notch signaling to the MSC-mediated induction of Treg cells. Here, we demonstrate that the Notch1 pathway is involved in MSC induction of a Treg-cell population.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

MSC/CD4+cocultures significantly increased the percentage of CD4+CD25high (5.28 ± 0.44% versus 1.65 ± 0.36% p < 0.001), CD4+FOXP3+ (9.16 ± 3.09% versus 3.6 ± 0.95% p < 0.05), CD25+FOXP3+ (7.49 ± 2.6% versus 2.16 ± 0.0775% p < 0.001), and CD4+CD25highGITR+ cells (34.01 ± 5.2% versus 11.5 ± 4.5% p < 0.05) compared with CD4+ control cells (Supporting Information Fig. 1). FOXP3 mRNA expression was increased 3.8 ± 0.92-fold (p < 0.01). CD4+CD25+-enriched cells significantly reduced CD4+CD25 cell proliferation by up to 83 ± 18%.

As MSC/CD4+cells cocultures induced a Treg-cell population with suppressive capacity we then investigated whether the Notch1 pathway was involved in the induction of Treg cells by MSCs. MSCs expressed the Notch1 ligands Jagged1, Jagged2, and DLL 1,3–4 mRNAs, with Jagged1 being prevalent (Fig. 1A). Western blot analysis confirmed Jagged1 expression (Fig. 1B and Supporting Information Fig. 4) After a 4-day CD4+ cell coculture with MSCs we analyzed whether Notch1 signaling was activated by assessing expression of Notch1, its downstream target HES1, and the negative regulator FBW7. Compared with control CD4+ cells, Notch1 and HES1-mRNA expression increased 2.07 ± 0.94- and 4.65 ± 2.7-fold, while FBW7 decreased by up to 0.5 ± 0.21-fold (Fig. 1C). Western blot analysis revealed significant over-expression of the Notch1 intracellular (IC) domain and the transmembrane/cytoplasmic portion (Fig. 1E). These data clearly demonstrated the Notch1 pathway was activated in CD4+ cells that were cocultured with MSCs and excluded potential autocrine Notch signaling in CD4+ cells that was able to induce Treg cells.

image

Figure 1. Activation and modulation of the Notch1 signaling pathway in CD4+ cell/MSC cocultures. (A) qRT-PCR analysis of the indicated Notch1 ligand levels in MSCs is shown. Data are shown as mean of N = 3 and are representative of three experiments performed. (B) Western blot analysis of Jagged1 expression in MSCs. GAPDH was used as loading control. Data shown are representative of three experiments performed. (C) qRT-PCR analysis of Notch1, HES1, and FBW7 mRNA expression in CD4+ cells after 4 days in culture with or without MSCs. Data are shown as mean + SD of N = 6 samples representative of six experiments performed. (D) qRT-PCR analysis of FOXP3, Notch1, HES1, and FBW7 mRNA expression in enriched iTreg cells, compared with that of non-Treg cells. Data are shown as mean + SD of N = 4 and representative of four experiments performed. (E) (i) Representative western blot of Notch1-TM and IC protein expression in CD4+ cells after 4 days in culture with or without MSCs. (ii) Absolute Notch1-TM and Notch1-IC amounts were calculated from samples pooled from four different experiments. Data are shown as mean + SD. Densitometry units (U) were calculated relative to GAPDH using Quantity One software. (F) qRT-PCR analysis of HES1 and FBW7 mRNA, from CD4+ cells after 4 days in coculture with MSCs in the presence of 2.5 μM GSI-I, or 10 μg/ml Notch1 neutralizing antibody (α-Notch1), or 5 μg/ml TGF-β1 neutralizing antibody (α-TGB-β1). DMSO and isotype antibodies were used as controls (CTRL). The data were normalized to GAPDH and shown as fold-change with respect to levels of control cells; shown as mean + SD of N = 5; and representative of five experiments performed (G-I). Western blot (i) and densitometric (ii) quantification of Notch1-TM and Notch1-IC expression in CD4+ cells after 4 days in culture with MSCs in the presence of (G) GSI-I, (H) Notch1 nutralizing antibody, or (I) TGF-β1 neutralizing antibody are shown. DMSO and isotype antibodies were used as CTRL. All data are shown as mean + SD of data pooled from at least three different experiments.

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After CD4+ cell-MSC cocultures, iTreg cells were purified by CD25 cell enrichment. Compared with CD25 negative cells (non-Treg cells), the Notch1 signaling was upregulated in the iTreg cells, the FOXP3 mRNA expression was doubled, Notch1 and HES1 were upregulated, and FBW7 was significantly downregulated, suggesting the MSC effects are specific to Treg cells (Fig. 1D).

In order to assess the Notch1 signaling contribution to MSCs-related Treg-cell induction, we used GSI-I to block the Notch1 pathway. GSI-I modulates Notch1-mediated induction of target genes by blocking Notch1-IC proteolysis. GSI-I treatment of cocultured CD4+/MSCs reduced HES1–mRNA upregulation by approximately 50% and increased FBW7 3.6 ± 1.6-fold, compared with controls (Fig. 1F). Western blotting showed downregulation of Notch1-IC and TM expression, confirming GSI-I inhibited MSC-mediated Notch1 activation (Fig. 1G). Compared with DMSO-treated controls, GSI-I significantly reduced the percentages of CD4+CD25+(5.7 ± 5% versus 11.9 ± 2.7% p < 0.05); CD4+CD25high (0.4 ± 0.5% versus 6.2 ± 2.6% p < 0.01); CD4+FOXP3+(3.5 ± 1.5% versus 9.3 ± 0.8% p < 0.001); CD25+FOXP3+ (5.99 ± 0.78% versus 1.40 ± 0.99% p < 0.001); CD4+CD25+FOXP3+ (22.6 ± 24.3% versus 69.5 ± 8.1% p < 0.05); and CD4+CD25highFOXP3+ (28.3 ± 17.38% versus 80.8 ± 11% p < 0.001) (Fig. 2A–E). FOXP3 expression was also significantly reduced to one-third of the control level in mRNA (Fig. 2F). The GSI-I effects were specific to CD4+ cells only, since the mRNA expression of Notch1 and Notch1-ligands on MSCs was not inhibited by GSI-I treatment (Supporting Information Fig. 3). Moreover, GSI-I treatment did not seem to modify Notch1 expression on MSCs in our system (data not shown).

image

Figure 2. Inhibition of the Notch1 signaling in CD4+ cell/MSC cocultures. (A–E) Representative plots (i) showing the percentage of (A) CD4+CD25+, CD4+CD25high, (B) CD4+FOXP3+, (C) CD25+FOXP3+, (D) CD4+CD25+FOXP3+, (E) CD4+CD25highFOXP3+ cells after 4 day CD4+ cell/MSC coculture in the presence of GSI-I or Notch1 neutralizing antibody (α-Notch1). DMSO and isotype antibody were used as CTRL). (ii, iii) The flow cytometric data are shown as mean + SD of data pooled from at least four different experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Data were analyzed by unpaired t-test using Prism3 Software (GraphPad Software). (F) qRT-PCR analysis of FOXP3 mRNA from CD4+ cell/MSC cocultures in the presence of GSI-I or Notch1 neutralizing antibody. DMSO and isotype antibody were used as CTRL. The data were normalized to GAPDH and shown as fold-change with respect to levels of control cells, shown as mean + SD of N = 5, and representative of five experiments performed. (G) Suppression assay of enriched iTreg cells obtained after CD4+ cell/MSC cocultures, alone and/or in the presence of GSI-I or anti-Notch1 antibody. Data are shown as percent inhibition and mean + SD of N = 3, representative of three experiments performed *p < 0.05. Data were analyzed by unpaired t-test using Prism3 Software (GraphPad Software).

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In a different set of experiments designed to exclude any GSI-I-mediated off-target effect, we used a Notch1 neutralizing antibody to block Notch1 signaling. Compared with IgG2b-treated control cells, HES1-mRNA expression was reduced 0.39 ± 0.22-fold and FBW7 was increased 2.7 ± 1.8-fold (Fig. 1F). Western blotting showed significant reductions in Notch1-IC and TM expression (Fig. 1H). Flow cytometry detected significant reductions in the percentages of CD4+CD25high Treg cells (8.41 ± 0.6% versus 6.09 ± 0.2% p < 0.05); CD4+FOXP3+ (3.6 ± 0.38% versus 7.48 ± 0.78% p < 0.01); CD25+FOXP3+ (5.19 ± 0.27% versus 2.24 ± 0.098% p < 0.01); CD4+CD25+FOXP3+ (46.59 ± 5.9% versus 81.9 ± 7.3% p < 0.01); and CD4+CD25highFOXP3+ (46.3 ± 4.6% versus 82.37 ± 5.4% p < 0.01) (Fig. 2A–E). FOXP3-mRNA expression was significantly reduced to half the control level (Fig. 2F). Altogether these data demonstrated that Notch1 pathway disruption by GSI-I and the anti-Notch1 antibody impaired MSC recruitment of Treg cells.

To study whether the Notch1 pathway was critical for MSC-recruitment of Treg cells, we performed a suppression assay in presence of either GSI-I or anti-Notch1 antibody. The suppressive capacity of CD25 enriched iTreg cells was significantly reduced after GSI-I and anti-Notch1 treatments (51 ± 7% and 49.7 ± 12.4% versus 83 ± 18% untreated cells) (Fig. 2G).

Interestingly, although Liotta et al. described Jagged1 involvement in MSC- suppression of T-cell proliferation [15], the elicited Treg-cell population and its fine regulation by Notch1/Jagged1 axis remained undiscovered. Here, we not only demonstrated for the first time that Notch1/Jagged1 signaling was involved in human MSCs-mediated Treg-cell induction but we also identified the Notch1 pathway as yet another mechanism in the development of iTreg cells.

The present experiments also demonstrated that FOXP3 was a downstream target of Notch1 signaling in human cells, thus extending data that had been obtained in murine models [16].

In our model, MSCs appear to regulate a fine inverse pattern of FBW7 and HES1 mRNAs. FBW7 has been shown to regulate HES1 and DELTEX1 gene expression to target the Notch1 pathway [17]. We hypothesize that Notch1 pathway members may interact with unknown MSC factors to influence FBW7 transcriptional regulation.

Previous studies demonstrated involvement of TGF-β1 production in MSCs-mediated Treg-cell induction [4, 18] and TGF-β1-Notch1 crosstalk [16] in peripheral Treg-cell maintenance. In our experiments, we quantified TGF-β1 expression in MSC supernatant as 293.3 ± 54.8 pg/ml. Blocking MSC-produced TGF-β1 did not change the percentage of iTreg cells (Supporting Information Fig. 2) or Notch1 TM and IC expression (Fig. 1I). FOXP3 (Supporting Information Fig. 2D), HES1, and FBW7 mRNA expression was also unchanged (Fig. 1F). Consequently, TGF-β1 did not play a role in MSC-mediated Treg-cell induction and there was no TGF-β-Notch1 crosstalk. Lack of T-cell receptor stimulation in our system may account for the discrepancy with other reports [19-21].

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Treg-cell function and its impairment with Notch1, and to a lesser extent, Jagged1, blockade was already demonstrated [22]. We found this machinery was also functional in Treg-cell recruitment by MSCs. Driving CD4+ T cells toward Treg-cell differentiation by coculture with MSCs might constitute a new advance in the use of Treg cells in clinical settings. More importantly, these findings indicate that Notch signaling activation reversed the otherwise unstable regulatory/suppressive properties of iTreg cells, ensuring sustained FOXP3 expression and more stable Treg-cell phenotypes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Cell isolation and MSC/T-cell cocultures

MSCs were obtained from healthy donor bone marrow [9]. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) using anti-CD4 microbeads (Miltenyi Biotec) and cocultured with MSCs for 4 days (ratio 5:1) [9]. In all present experiments, MSCs were allogeneic to the CD4+ cells employed for MSC/T-cell cultures and for the suppression assays. Informed consent was obtained from MSC and CD4+ cell donors. iTreg cells were enriched using anti-CD25 microbeads (Miltenyi Biotec). T cells were analyzed using MoAbs directed against CD45, CD3, CD4, CD25, (Coulter Corporation), GITR (Miltenyi Biotec), and FOXP3 with mouse IgG2-APC as isotype control (eBioscience) by CYTOMICS FC500 Cytometer (Coulter Corporation).

MSCs/CD4+ cells were cultured with 5 μg/ml anti-TGF-β1 (R&D system) with isotype-control mouse IgG1(R&D) as control; or 2.5 μM GSI-I (γ-secretase inhibitor I; Calbiochem) with DMSO as control; or 10 μg/ml anti-Notch1 (clone A6, lab Vision/neomarkers) with isotype-control mouse IgG2b (R&D) as control. After MSCs/CD4+ cell cocultures iTregs were isolated by CD25 microbeads and added to conventional T cells in a 96-h suppression assay (ratio between conventional T cells and Treg cells was 1 : 0.1) as previously described [23, 24].

To exclude MSC contamination, CD45 and CD90 staining was performed before iTreg cells were used for further experiments.

Notch1 signaling analysis

Quantitative real time PCR was performed using the 7900Ht Fast Real Time PCR System (Applied Biosystems) with SYBR Green kits (Applied Biosystems). For a record of primers, see Supporting Information Table 1. Western blot analysis was performed using anti-Notch1 (clone bTAN20), anti-Jagged1 (clone TS1.15H) (DSHB), and anti-GAPDH (Sigma-Aldrich) antibodies [25].

Statistics

Data were analyzed by unpaired two-tailed t-tests using Prism3 software (GraphPad Software).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

We would like to thank Dr. Geraldine Anne Boyd for her help. This work was supported by Associazione Umbra Leucemie e Linfomi, Perugia, Italy and Associazione Italiana Leucemie, Linfomi e Mieloma, L'Aquila Section, L'Aquila, Italy.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
IC

intracellular

iTreg

inducible Treg

MSC

mesenchymal stem cell

TM

transmembrane

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

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Supporting Information Table 1. List of primers used for qRT-PCR.

Supporting Information Figure 1. MSC recruitment of Treg cells. (Ai-Ci) Representative plots showing the percentage of CD4+CD25high, CD4+FOXP3+,CD25+FOXP3+ cells after 4 day's CD4+/MSCs co-culture in the absence or presence of MSCs. (Aii-Bii) Graphic representation of flow cytometric data derived from at least five independent experiments (mean ± SD) *p < 0.05, **p < 0.01 ***p < 0.001. (D) qRT-PCR analysis of FOXP3 mRNA, was analysed from cells prepared under the same conditions as in panel Ai. The data was normalized to GAPDH and represented as fold change with respect to levels of control cells. Data derived from five independent experiments (mean ± SD); **p < 0.01. (E) Graphic representation of flow cytometric GITR expression after 4 day's CD4+/MSCs co-culture in the absence or presence of MSCs, data derived from four independent experiments (mean ± SD)*p < 0.05 Data were analyzed by unpaired t-test using Prism3 Software (GraphPad Software).

Supporting Information Figure 2. TGF-®1 is not involved in MSC-mediated Treg cells induction. (Ai-Ci) Representative plots showing the percentage of CD4+CD25+, CD4+CD25high, CD4+FOXP3+, CD4+CD25+FOXP3+ and CD4+CD25highFOXP3+ cells after 4 day's CD4+/MSCs co-culture in presence of TGF-®1 neutralizing antibody. Isotype antibody was used as controls (CTRL). (Aii-Cii) Graphic representation of flow cytometric data derived from at least three independent experiments (mean ± SD). (D) qRT-PCR analysis of FOXP3 mRNA, was analysed from CD4/MSCs co-cultures in the presence of TGF−®1 neutralizing antibody. Isotype antibody was used as controls (CTRL). The data was normalized to GAPDH and represented as fold change with respect to levels of control cells. Data derived from at least four independent experiments (mean ± SD).

Supporting Information Figure 3. GSI-I treatment no effect on MSC-Notch signaling pathway. (A) qRT-PCR analysis of Notch1, and Notch1-ligands mRNA (Jagged1, Jagged2, DLL1, DLL3, DLL4) on MSCs treated with GSI-I compared with untreated MSCs as control. The data was normalized to GAPDH and represented as fold change with respect to levels of control cells Data derived from three independent experiments (mean ± SD).

Supporting Information Figure 4 Western blot assays of Jagged1 expression on MSCs. Representative western blot analysis of Jagged1 expression in MSCs. GAPDH was used as loading control.

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