IDO and CD40 May Be Key Molecules for Immunomodulatory Capacity of the Primed Tonsil-Derived Mesenchymal Stem Cells

Abstract

Background: Tonsil-derived mesenchymal stem cells (T-MSCs) were reported to have suppressive effect on T cells, yet much remains unknown about the underlying mechanisms supporting this effect. We investigated the underlying mechanism of the immunomodulatory effect of T-MSCs on immune cell proliferation and cytokine production. Methods: We isolated T-MSCs from human palatine tonsil and evaluated the immunomodulatory capacity using RT-PCR, ELISA, and flow cytometry. Additionally, we assessed the expression of various soluble factors and several costimulatory molecules to detect the priming effect on T-MSCs. Results: T-MSCs significantly inhibited the immune cell proliferation and cytokine expression (TNF-α and IFN-γ) in the direct co-culture, but there was no suppressive effect in indirect co-culture. Additionally, we detected a remarkably higher expression of indoleamine 2,3-dioxygenase (IDO) in the primed T-MSCs having co-expression CD40. Moreover, immune cells or CD4⁺ T cells showed lower TNF-α, IFN-γ, and IL-4 expression when the primed T-MSC were added; whereas those findings were reversed when the inhibitor for IDO (not IL-4) or CD40 were added. Furthermore, T-bet and GATA3 levels were significantly decreased in the co-cultures of the primed T-MSCs and CD4⁺ T cells; whereas those findings were reversed when we added the neutralizing anti-CD40 antibody. Conclusions: Primed T-MSCs expressing IDO and CD40 may have immunomodulatory capacity via Th1-mediated and Th2-mediated immune response.

Keywords: CD40; indoleamine 2,3-dioxygenase; mesenchymal stem cell; tonsil.

Figure 1

Characterization and immunosuppressive activity of tonsil-derived mesenchymal stem cells (T-MSCs). (A) T-MSCs are characterized for marker expression by flow cytometry. Dashed histograms indicate staining with isotype control antibodies and solid histograms denote the specific expression of each indicated marker. In vitro immunosuppressive activity of T-MSCs according to different concentrations (absence, 5 × 10⁴, 1 × 10⁵, and 2 × 10⁵) on (B) CD4⁺ T cell and (C), whole immune cells are determined by CFSE assay. Dot blots are representative of each independent experiment and the graphic data is the ± SEM from each independent experiment (* p < 0.05 and ** p < 0.001). T-MSC: tonsil-derived mesenchymal stem cell; PHA, phytohemagglutinin; CC, cell–cell contact; TW, transwell.

Figure 2

Assessment of tonsil-derived mesenchymal stem cells (T-MSCs)-mediated immunosuppressive function. (A) Flow cytometric analysis of the proliferating immune cells after direct or indirect co-cultured with T-MSCs for 4 days. (B) Flow cytometric analysis of TNF-α, IFN-γ, IL-17A, and IL-4 expression in CD4⁺ T cells after direct or indirect co-cultured with T-MSCs for 4 days. Dot blots are representative of each independent experiment and the graphic data is the ± SEM from three independent experiments (* p < 0.05). T-MSC: tonsil-derived mesenchymal stem cell; PHA, phytohemagglutinin; CC, cell–cell contact; TW, transwell.

Figure 3

Effect of priming on the production of critical immunomodulatory factors in tonsil-derived mesenchymal stem cell (T-MSC). (A) Comparison of soluble factors expression, including IDO, COX-2, HO-1, TGF-β1, IL-10, and iNOS between T-MSC^Con and T-MSC^Primed using RT-PCR (* p < 0.05, ** p < 0.001, *** p = 0.0008). (B) Comparison of costimulatory molecule expression, such as PD-L1, ICOSL, and iNOS between T-MSC^Con and T-MSC^Primed using flow cytometry. (C) IDO inductivity of T-MSC^Primed is confirmed by ELISA (*** p = 0.0004). (D) Protein expression of CD40 and IDO is confirmed by flow cytometry between T-MSC^Con and T-MSC^Primed. T-MSC^Con, tonsil-derived mesenchymal stem cell with the addition of vehicle control; T-MSC^Primed, tonsil-derived mesenchymal stem cell primed with TNF-α and IFN-γ; IDO, indoleamine 2,3-dioxygenase quantification; HO-1, heme oxygenase; PD-L1, programmed death-ligand 1; ICOSL, inducible T cell costimulator ligand.

Figure 4

Indoleamine 2,3-dioxygenase quantification (IDO) and CD40 are key molecules on the immunomodulatory effect of the primed tonsil-derived mesenchymal stem cells (T-MSCs). (A) Expression of TNF-α, IFN-γ, and IL-4 in CD4⁺ T cells according to the different groups (control, PHA, T-MSC^Con, T-MSC^Primed and inhibitor IDO) (B) Expression of TNF-α, IFN-γ, and IL-4 in CD4⁺ T cells according to the different groups (control, TCR, T-MSC^Con, T-MSC^Primed, and Anti-CD40Ab) (C) Expression level of transcription factors of Th1 (T-bet) and Th2 (GATA3) both in the co-cultures of T-MSCs and CD4⁺ T cells according to the different groups (control, TCR, T-MSC^Con, T-MSC^Primed, and Anti-CD40Ab). Dot blots are representative of each independent experiment. The graphic data is the ± SEM from each independent experiment. * p < 0.05 and ** p < 0.01. T-MSC^Con, tonsil-derived mesenchymal stem cell with the addition of vehicle control; T-MSC^Primed, tonsil-derived mesenchymal stem cell primed with TNF-α and IFN-γ; Anti-CD40Ab, Neutralizing anti-CD40 antibodies.

Figure 4

Indoleamine 2,3-dioxygenase quantification (IDO) and CD40 are key molecules on the immunomodulatory effect of the primed tonsil-derived mesenchymal stem cells (T-MSCs). (A) Expression of TNF-α, IFN-γ, and IL-4 in CD4⁺ T cells according to the different groups (control, PHA, T-MSC^Con, T-MSC^Primed and inhibitor IDO) (B) Expression of TNF-α, IFN-γ, and IL-4 in CD4⁺ T cells according to the different groups (control, TCR, T-MSC^Con, T-MSC^Primed, and Anti-CD40Ab) (C) Expression level of transcription factors of Th1 (T-bet) and Th2 (GATA3) both in the co-cultures of T-MSCs and CD4⁺ T cells according to the different groups (control, TCR, T-MSC^Con, T-MSC^Primed, and Anti-CD40Ab). Dot blots are representative of each independent experiment. The graphic data is the ± SEM from each independent experiment. * p < 0.05 and ** p < 0.01. T-MSC^Con, tonsil-derived mesenchymal stem cell with the addition of vehicle control; T-MSC^Primed, tonsil-derived mesenchymal stem cell primed with TNF-α and IFN-γ; Anti-CD40Ab, Neutralizing anti-CD40 antibodies.