Figure 3. Harmine's effects on canonical T_reg/Th17 pathways.

All data representative of at least 2 independent experiments. (A) Effect of harmine on murine T_reg, Th1 and Th17 differentiation. (B) Effect of harmine on absolute numbers of total live and T_reg cells. (C) Effect of harmine on human T_reg differentiation. (D) Effect of harmine on Th differentiation under Th0 conditions as shown by percentage of cells with indicated markers. (E) Harmine's pro-T_reg effect when added or removed at different times after T_reg^low stimulation as shown by percentage of maximal T_reg enhancement (% max T_reg enh). (F and G) Volcano plots comparing p value vs fold change in gene expression in naïve CD4⁺ T cells, T_reg^hi-T_reg cells and T_reg^HAR-T_reg cells as indicated. Previously reported signature genes for T_reg cells (F) and activated T cells (G) are highlighted in red (upregulated) and green (downregulated). Numbers on the right and left reflect genes that are up- and down-regulated in the indicated comparison respectively with χ² test p values in the middle. (H) Median fluorescence intensity (MFI) of FOXP3 in T_reg cells generated under indicated conditions. (I) Time-course analysis of FOXP3 expression in cells cultured under indicated conditions. (J and K) Western blot analyses showing effect of T_reg enhancers on S6 kinase, SMAD2 and SMAD3 phosphorylation when added under T_reg^low conditions. Numbers denote fractional phosphorylation relative to T_reg^low conditions. (L) Effect of harmine (blue) on RORγT expression and STAT3 phosphorylation in Th17^hi conditions (gray). (M) qPCR analyses showing effects of harmine on key Th17 genes at 4 days (left) and 2 hours (right) after stimulation. Gray and blue bars represent Th17^hi and Th17^hi + harmine conditions, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, Student's t-test with Holm-Sidak correction (B, C, D, M). See also Figure 3—figure supplements 1–5.

DOI: http://dx.doi.org/10.7554/eLife.05920.015

Figure 3—figure supplement 1. Effect of harmine on cellular proliferation.

CFSE-labelled CD4⁺ T cells were stimulated under T_reg^low conditions and proliferation assessed daily. The effect of adding harmine, rapamycin, or high TGFβ (T_reg^hi) was compared as shown.

DOI: http://dx.doi.org/10.7554/eLife.05920.016

Figure 3—figure supplement 2. Effect of harmine on absolute numbers of Th17 cells.

***p < 0.001, Student's t-test.

DOI: http://dx.doi.org/10.7554/eLife.05920.017

Figure 3—figure supplement 3. Effect of addition or removal of harmine at different times on Th17 differentiation.

DOI: http://dx.doi.org/10.7554/eLife.05920.018

Figure 3—figure supplement 4. Correlation between gene expression in T_reg^hi-T_reg cells and T_reg^HAR-T_reg cells, relative to naïve CD4⁺ T cells.

DOI: http://dx.doi.org/10.7554/eLife.05920.019

Figure 3—figure supplement 5. Qualitative analyses of genomewide expression in T_reg^HAR-T_reg cells.

Previously described methods were used to identify similarities between T_reg^HAR-T_reg cells and other specialized T_reg subsets (Joller et al., 2014). Volcano plots compare relative expression of genes in T_reg^hi-T_reg cells and T_reg^HAR-T_reg cells. Overlaid are genes of previously identified signatures; red and green reflect genes up- and down-regulated in these signatures respectively. Numbers on the right and left reflect genes that are up- and down-regulated in the indicated comparison respectively with χ² test p values in the middle.

DOI: http://dx.doi.org/10.7554/eLife.05920.020