Notch1 modulates mesenchymal stem cells mediated regulatory T‐cell induction

Abstract

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 CD4can be recruited by MSCs from CD4 + T cells either indirectly, 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 dioxygenase (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]. * These authors contributed equally to this work. Results and discussionMSC/CD4 + cocultures significantly increased the percentage of CD4 + CD25 high (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 + CD25 high GITR + 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.After CD4 + cell-MSC cocultures, iTreg cells were purified by CD25 cell enrichment. Compared with CD25 negative cells (nonTreg 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 MSCsrelated Treg-cell induction, we used GS...

Regulation of innate and adaptive immunity by Notch

“…Accordingly, in vivo treatment of mice with γ-secretase inhibitors reduced the TGFβ-mediated induction of FOXP3, decreased peripherally induced T Reg cell development and maintenance, and led to the development of auto immune hepatitis 5 . The importance of Notch signalling in ensuring sustained FOXP3 expression and maintenance of peripherally induced T Reg cells was recently confirmed in human cells 98 . Pluripotent stem cells transduced with FOXP3 and co-cultured on Notch ligandexpressing stromal cells generated stable T Reg cells in vitro 99 , suggesting that this could be a strategy for producing stable T Reg cells for therapeutic adoptive transfer.…”

Mesenchymal Stromal Cells Protect Against Caspase 3-Mediated Apoptosis of CD19+Peripheral B Cells Through Contact-Dependent Upregulation of VEGF

“…Over the last number of years, the importance of the notch signalling pathway in immune modulation by MSC has been clearly identified. The roles for notch signalling in MSC modulation of DC maturation and antigen presentation [31] and in the induction of regulatory T cells [32,33] have now been established. Therefore it was hypothesised that notch signalling between MSC and CD19 + B cells increased peripheral B cell viability.…”

Delta-like 1–Mediated Notch Signaling Enhances the In Vitro Conversion of Human Memory CD4 T Cells into FOXP3-Expressing Regulatory T Cells

“…A role for Notch signaling in human Treg development and expansion has been previously suggested (20,21,23). In fact, human cord blood CD34 + cells differentiate into mature Treg upon coculture with OP9-DL1, suggesting that Notch is involved in human thymic Treg development (21).…”

“…Moreover, APCs overexpressing human Jag1 promoted the expansion of alloantigenspecific cells with regulatory properties from human naive CD4 cells (20). Additionally, mesenchymal stem cells cultivated with human CD4 + T cells enhanced the recovery of FOXP3 + cells via a Notch1-mediated mechanism (23). However, these studies did not discriminate whether Notch signaling was promoting Treg expansion and/or Treg de novo induction.…”

“…There is solid experimental data in mice demonstrating a crucial role for Notch signaling in the thymic generation of Treg, their peripheral expansion and function, as well as in the in vitro conversion of conventional T cells into iTreg (15)(16)(17)(18)(19). In humans, there is evidence suggesting a role of Notch signaling in thymic Treg development as well as in peripheral Treg expansion (20)(21)(22)(23), although the putative role of Notch signaling in iTreg conversion has never been formally assessed.…”