Mesenchymal stromal cells inhibit CD25 expression via the mTOR pathway to potentiate T-cell suppression

Abstract

Mesenchymal stromal cells (MSCs) are known to suppress T-cell activation and proliferation. Several studies have reported that MSCs suppress CD25 expression in T cells. However, the molecular mechanism underlying MSC-mediated suppression of CD25 expression has not been fully examined. Here, we investigated the mTOR pathway, which is involved in CD25 expression in T cells. We showed that MSCs inhibited CD25 expression, which was restored in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Since CD25 mRNA expression was not inhibited, we focused on determining whether MSCs modulated components of the mTOR pathway in T cells. MSCs increased the phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) and decreased the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, the expression of 4E-BP1 increased dramatically in the presence of MSCs. An m⁷GTP pull-down assay showed increased binding of 4E-BP1 to the 5' cap-binding eukaryotic translation initiation factor 4E (eIF4E) complex in the presence of MSCs, which resulted in inhibition of mRNA translation. Treatment with 4EGI-1, a synthetic inhibitor of mRNA translation, also reduced CD25 expression in T cells. Polysome analysis confirmed decreased CD25 mRNA in the polysome-rich fraction in the presence of MSCs. Taken together, our results showed that nitric oxide, produced by MSCs, inhibits CD25 translation through regulation of the LKB1-AMPK-mTOR pathway to suppress T cells.

Figure 1

Inhibition of T-cell CD25 expression by MSCs. Lymphocytes from the spleen and lymph nodes were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of MSCs. (a) Lymphocytes (2 × 10⁵) were cultured for 3 days. During the final 16 h of culture, 1 μCi ³H-thymidine was added, and T-cell proliferation was determined by thymidine incorporation. The number of MSCs used was 1/100, 1/50, and 1/20 that of lymphocytes. (b) Lymphocytes (1 × 10⁶) were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of MSCs. IL-2 expression in the cell culture media was measured by ELISA. The number of MSCs used was 1/20 that of lymphocytes. (c) IL-2 mRNA expression was measured at different time points by qRT-PCR. Targets were normalized to 18S ribosome levels. (d) CD25 protein expression was measured by flow cytometry. (e) Intra- and extracellular CD25 expression was measured at 48 h by flow cytometry. (f) sCD25 protein expression in cell culture media was measured by ELISA. (g) CD25 mRNA expression was measured by qRT-PCR. Targets were normalized to 18S ribosome levels. Similar results were obtained in three independent experiments. −: unstimulated, +: anti-CD3 and anti-CD28 antibody-stimulated, M: MSCs. *P<0.05, **P<0.01, n.s., not significant, n.d., not detected

Figure 2

Inhibition of CD25 expression by NO. Lymphocytes were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of MSCs. (a) Cells were cultured for 3 days. During the final 16 h of culture, 1 μCi ³H-thymidine was added. T-cell proliferation was determined by thymidine incorporation. 1-MT: IDO inhibitor, L-NMMA: iNOS inhibitor. (b) Lymphocytes (1 × 10⁶) were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of MSCs. CD25 protein expression was measured at 48 h by flow cytometry. (c) CD25 mRNA expression was measured at 48 h by RT-PCR. Target levels were normalized to GAPDH levels. (d) IL-2 and NO levels in cell culture media were measured at 48 h by ELISA. (e) Intracellular phospho-STAT5 was detected at 48 h by flow cytometry. (f) Cells were cultured with either MSCs or the NO donor, DEANO. CD25 protein expression was measured at 48 h by flow cytometry. (g) IL-2 was detected at 48 h by ELISA. Similar results were obtained in two (e–g) or three (a–d) independent experiments. iso: isotype control;−: unstimulated, +: anti-CD3 and anti-CD28 antibody-stimulated; M: MSCs, MI: MSCs + L-NMMA, D: DEANO, ***P<0.001, **P<0.01 compared to the controls

Figure 3

Arginine is a source of NO. Lymphocytes were stimulated with anti-CD3 and anti-CD28 antibodies for 48 h in the presence or absence of MSCs. Arginine-free RPMI 1640 media were used. (a) CD25 expression was detected in CD4⁺ and CD8⁺ T cells by flow cytometry. (b) IL-2 and NO were measured by ELISA in the cell culture media. Similar results were obtained in two independent experiments. W, with arginine, W/O, without arginine, −: unstimulated, +: anti-CD3 and anti-CD28 antibody-stimulated; M, MSCs; **P<0.01 compared to the controls

Figure 4

Effect of IFN-γRα and iNOS knockdown MSCs on CD25 expression. To knock down IFN-γRα, MSCs were infected with shRNA-harboring lentiviral particles (a–f). IFN-γRα KD MSCs were stimulated with IFN-γ (20 ng/ml) and TNF-α (10 ng/ml). IFN-γRα and iNOS expression was measured after 24 h by (a) RT-PCR or (b) western blotting. IFN-γRα KD MSCs were cultured with lymphocytes for 48 h. (c) NO and (d) T-cell proliferation were measured by ELISA and thymidine incorporation, respectively. (e) CD25 cell surface expression was measured at 48 h by flow cytometry. (f) IL-2 secretion into cell culture media was measured at 48 h by ELISA. To knock down iNOS, MSCs were transfected with siRNA (g–k). iNOS KD MSCs were stimulated with IFN-γ (20 ng/ml) and TNF-α (10 ng/ml). iNOS mRNA and protein were measured after 24 h by (g) qRT-PCR or (h) western blotting. iNOS KD MSCs were cultured with lymphocytes for 48 h. (i) NO and (j) CD25 cell surface expression were measured by ELISA and flow cytometry, respectively. (k) IL-2 level in culture media was measured at 48 h by ELISA. Similar results were obtained in two independent experiments. WT, wild type; KD, knockdown; I, IFN-γ, T; TNF-α; M, MSCs, *P<0.05, **P<0.01 compared to the controls

Figure 5

MSCs inhibit mTOR signaling. (a) T cells were cultured with MSCs for 24 h. T cells were stimulated for 12 h, after which DEANO was added and the cells were cultured for 2 h. Phosphorylation of LKB1 and AMPKα was detected by western blotting. (b) T cells were cultured with MSCs for 24 h in the presence or absence of L-NMMA. Phosphorylation of S6K and 4E-BP1 was measured by western blotting. (c) 4E-BP1 mRNA was measured by qRT-PCR. Target levels were normalized to 18S ribosome levels. (d) Phosphorylation of 4E-BP1 was measured by western blotting. (e) To measure 4E-BP1 binding to eIF4E, an m⁷GTP pull-down assay was performed. eIF4E-binding to 4E-BP1 was detected by western blotting. (f) T cells were cultured with MSCs for 24 h. Polysome-rich RNA was separated from total RNA using a 15–55% sucrose gradient system. CD25 mRNA was detected by qRT-PCR. Target levels were normalized to 18S ribosome levels. Similar results were obtained in two (c and e) and three (a, b, d and f) independent experiments. −: unstimulated, +: anti-CD3 and anti-CD28 antibody-stimulated, M: MSCs, MI: MSCs + L-NMMA, D: DEANO, NS: not significant, *P<0.05, **P<0.01 compared to the controls

Figure 6

Inhibition of CD25 and CD122 protein expression by mRNA translation inhibitor 4EGI-1. Lymphocytes were stimulated in the presence of 4EGI-1. (a) CD122 and CD132 cell surface expression was measured at 40 h by flow cytometry. (b) Expression of CD25 and CD122 mRNA was measured at 20 h by qRT-PCR. Target levels were normalized to GAPDH levels. (c) IL-2 expression was measured at 40 h by ELISA. Similar results were obtained in two independent experiments. 4E: 4EGI-1, *P<0.05 compared to the controls