Figure 1.
Western blot analysis of MAP kinase and Smad phosphorylation in CD4+CD25− and CD4+CD25+ T cells (2–4×106 cells).
A: Analysis of p38 and ERK2 expression as well as p38 phosphorylation in CD4+CD25− and CD4+CD25+ T cells freshly isolated from spleen of wild type mice or transgenic mice overexpressing a dominant negative TGF-beta type II receptor in T cells. B: Analysis of Smad 2/3, Smad 7, Foxp3, actin, p-Smad 2, p-Smad 3, p-JNK and p-ERK1/2 from CD4+CD25− and CD4+CD25+ T cells freshly isolated from spleen of wild type animals. C: Densitometry and ratio of phosphorylated to unphosphorylated p38 and Smad 2/3 (p<0.05). The experiments were at least repeated three times giving similar results.
Figure 2.
TGF-beta1 activates Smad 3 and p38 MAP kinase in CD4+CD25− T cells.
Freshly isolated CD4+CD25− T cells (4×106) were cultured with or without TGF-beta1 for 4 or 16 hours. Cells were activated with plate bound anti-CD3 mAb (2 µg/ml) and soluble anti-CD28 mAb (2 µg/ml) as indicated. Smad 3 and p38 activation were measured using Western blot. The experiments were repeated three times giving similar results.
Figure 3.
Signaling via p38 MAP kinase is required for the in vitro conversion of CD4+CD25− T cells into TGF-beta1-induced Foxp3+ Treg (Ti Treg).
CD4+CD25− T cells were activated with plate bound anti-CD3 mAb (2 µg/ml) and soluble anti-CD28 mAb (2 µg/ml) for 4 days in the presence or absence of TGF-beta1 (2 ng/ml). Kinase inhibitors were added every 12 h (SB203580 (10 µM), SP600125 (10 µM), PD89059 (50 µM)). A: Schematic experimental procedure. Ti Treg: TGF-beta-induced Treg. B: Foxp3-expression and in vitro suppressor assay using CD4+CD25− CFSE-labelled responder T cells isolated from spleen and suppressor T cells generated by in vitro conversion as described above. Cells were washed three times before adding to the culture. Responder and suppressor cells were added at the indicated ratios [×105]. Data are representative of three independent experiments using SP600125, PD89059, SB202190 and five independent experiments using SB203580.
Figure 4.
Converted cells express CD25 and Foxp3 and are functional in vitro.
A: Representative CD25-/Foxp3-expression after in vitro conversion into iTreg in the presence or absence of SB203580. B: In vitro suppressor assay using CD4+CD25− CFSE-labelled responder T cells isolated from spleen and suppressor T cells generated by in vitro conversion as described. Responder and suppressor cells were added at a ratio of 1∶1. Suppressor cells were washed three times before addition to the assay.
Figure 5.
p38 MAP kinase inhibitor SB203580 specifically inhibits the phosphorylation of p38 MAP kinase downstream target MAPKAP2.
A: CD4+CD25− T cells were cultured with TGF-beta1 (2 ng/ml) and SB203580 (10 µM), SP600125 (10 µM), or PD89059 (50 µM) for 45 min. Control cells were cultured without TGF-beta1. B: CD4+CD25− T cells were cultured for 4 hours with TGF-beta1 (2 ng/ml) and SB203580 (10 µM). P-MAPKAP2, p-ERK, p-c-jun, p-Smad 3 and actin were determined using Western blot. Results are representative of two independent experiments.
Figure 6.
P38 MAP kinase signaling is not required for the in vitro suppressive function of CD4+CD25+ T cells.
Freshly isolated CD4+CD25− T cells from spleen were labelled with CFSE and cultured for four days under CD3 stimulation (3 µg/ml) in the presence of allogeneic CD3− spleen cells with or without CD4+CD25+ T cells freshly isolated from wild type mice, added at the indicated cell numbers (responder+suppressor cells×105). DMSO or SB203580 (10 µM) were added to the culture twice daily as indicated. CFSE dilution was measured by flow cytometry after 4 days of coculture. Data are representative of four independent experiments.