Figure 5. Sodium Chloride Reverts the Suppressive Function of Naturally Occurring Treg Cells but Not TGF-β-Induced Treg Cells In vitro and In vivo.

(A) tTregs isolated from thymus and TGF-β-induced iTregs were stimulated with anti-CD3/28 microbeads (5 cells per bead), IL-2 (50 U/mL) in standard media, or with additional 40 mM NaCl for 72 h, followed by co-cultured with enriched T cells labeled with CFSE in the presence of soluble anti-CD3 (0.025 μg/mL). Baseline indicates no addition of conditioned Treg subsets. CFSE dilution was detected by FACS after 3 days.

(B) Histograms depict cellular suppression and are gated on CD8⁺ T cells (n = 5).

(C) tTregs and iTregs were prepared from CD90.1⁺ Foxp3⁺ GFP knock-in mice and some of them were pre-treated by sodium chloride (40 mM). D90.2⁺CD4⁺CD25⁻CD62L⁺CD44⁻ naive T cells alone or together with the various Treg subsets were adoptively transferred into Rag1^−/− mice via i.p. injection. All mice were sacrificed at 35 days after the cell transfer, weight loss was analyzed for disease severity (6 mice in each group in one experiment). Body weight of the recipient mice was presented as a percentage of the initial weight.

(D) Gross morphology of colons was shown.

(E) The loss of Foxp3-GFP of the four sets were also compared in mLN, cells were gated on CD90.1⁺ cell. Representative results (mean ± SEM) from three independent experiments are shown.

(F) The bar graph depicts a summary of the experiment (6 mice in each group in one experiment). Statistical analyses were performed using Student’s t test and one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.