Figure 6.
Acute inflammation due to Treg deficiency was reversed by an mRNA translation inhibitor. (A–C) Mouse naive CD4 T cells were isolated from the spleens of naive C57BL/6 mice and CFSE labeled. The cells were stimulated with equal number of anti-mouse CD3/CD28-conjugated beads in the presence of the indicated concentration of RocA, FK506, or vehicle for 24 h (A and C) or 72 h (B). The cells were stained for anti-CD4 (A and C) or anti-CD4 and anti-CD25 (B) for flow cytometric analysis. The data of frequency of CD25 in A was summarized in C. Murine IL-2 was assayed by ELISA from supernatants collected after 24 h after stimulation (C). (D) Incorporation of PMY analyzed by flow cytometry after RocA treatment. (E–G) Foxp3DTR mice were treated with DT every other day and 0.2 mg/kg of RocA everyday starting from 2 d before DT treatment, followed by isolation of splenocytes at day 8. The cells were stained with anti-CD4, anti-TCRβ, and anti-CD44 followed by restimulation with PMA and Ionophore for 6 h. The cells were stained by anti–IL-17A and anti-IFNγ, followed by flow cytometric analysis (E). The body weight of mice at day 8 (F). The number of Th1 cells (IFNγ+CD44hiCD4+TCR-β+) and pathogenic Th17 cells (IFNγ+IL-17A+CD44hiCD4+TCR-β+; G). (H) Human naive CD4 T cells isolated from frozen PBMC, subjected to CFSE labeling. The cells were stimulated with anti-human CD3 and anti-human CD28–conjugated beads in the presence of 10 nM of RocA or vehicle or without the beads indicated as unstim. After 3 d, cells were stained with anti-human CD25 and anti-CD4, subjected to flow cytometry to analyze the frequency of divided cells and CD25+ cells. (I) Human memory Th1 cells isolated as CD4+CD25−CD45RA−CXCR3+CCR6− cells from frozen PBMC were incubated in the presence of 10 nM of RocA or vehicle for 2 d, followed by restimulation with PMA and Ionophore for 6 h. The cells were surface stained by anti-CD4 and subjected to cytoplasmic staining for IFNγ and IL-13. The frequency of IFNγ+ cells is summarized in the dot plots. One-way ANOVA was used for C, D, and H; two-tailed t test (unpaired) was applied for F and G; two-tailed Mann–Whitney test (unpaired) was applied for I. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The above experiments were repeated at least twice.

Acute inflammation due to Treg deficiency was reversed by an mRNA translation inhibitor. (A–C) Mouse naive CD4 T cells were isolated from the spleens of naive C57BL/6 mice and CFSE labeled. The cells were stimulated with equal number of anti-mouse CD3/CD28-conjugated beads in the presence of the indicated concentration of RocA, FK506, or vehicle for 24 h (A and C) or 72 h (B). The cells were stained for anti-CD4 (A and C) or anti-CD4 and anti-CD25 (B) for flow cytometric analysis. The data of frequency of CD25 in A was summarized in C. Murine IL-2 was assayed by ELISA from supernatants collected after 24 h after stimulation (C). (D) Incorporation of PMY analyzed by flow cytometry after RocA treatment. (E–G) Foxp3DTR mice were treated with DT every other day and 0.2 mg/kg of RocA everyday starting from 2 d before DT treatment, followed by isolation of splenocytes at day 8. The cells were stained with anti-CD4, anti-TCRβ, and anti-CD44 followed by restimulation with PMA and Ionophore for 6 h. The cells were stained by anti–IL-17A and anti-IFNγ, followed by flow cytometric analysis (E). The body weight of mice at day 8 (F). The number of Th1 cells (IFNγ+CD44hiCD4+TCR-β+) and pathogenic Th17 cells (IFNγ+IL-17A+CD44hiCD4+TCR-β+; G). (H) Human naive CD4 T cells isolated from frozen PBMC, subjected to CFSE labeling. The cells were stimulated with anti-human CD3 and anti-human CD28–conjugated beads in the presence of 10 nM of RocA or vehicle or without the beads indicated as unstim. After 3 d, cells were stained with anti-human CD25 and anti-CD4, subjected to flow cytometry to analyze the frequency of divided cells and CD25+ cells. (I) Human memory Th1 cells isolated as CD4+CD25−CD45RA−CXCR3+CCR6− cells from frozen PBMC were incubated in the presence of 10 nM of RocA or vehicle for 2 d, followed by restimulation with PMA and Ionophore for 6 h. The cells were surface stained by anti-CD4 and subjected to cytoplasmic staining for IFNγ and IL-13. The frequency of IFNγ+ cells is summarized in the dot plots. One-way ANOVA was used for C, D, and H; two-tailed t test (unpaired) was applied for F and G; two-tailed Mann–Whitney test (unpaired) was applied for I. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The above experiments were repeated at least twice.

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