Figure 4: Nr4a2 induces suppression activity in vitro by upregulating CD25 and repressing IL-2.

(a) qPCR analysis of the effects of Nr4a2 expression on the expression of Treg effector genes. mRNA was prepared from naïve T cells (columns 1 and 2), Tregs (columns 3 and 4), the GFP⁺ fraction of eMIGR1 retrovirus-transduced cells (columns 5, 6, 11 and 12), and the GFP⁺Foxp3^– (columns 7, 8, 13 and 14) or GFP⁺Foxp3⁺ (columns 9, 10, 15 and 16) fraction of eMIGR1-Nr4a2 retrovirus-transduced cells, which were incubated with (even-numbered columns) or without (odd-numbered columns) PMA/ionomycine for 2 h. Retrovirus-transduced cells were sampled at 48 h (blue bars, columns 5 to 10) and 84 h (green bars, columns 11 to 16) after infection. Expression levels of the indicated mRNA, are presented relative to the Hprt expression. Data are representative of three independent experiments (mean and s.d. of triplicate). (b) In vitro suppression assay. Top: suppression of CFSE-labeled naïve CD4⁺ T cells (responder, Tresp) by Tregs (open squares), the GFP⁺ fraction of eMIGR1 retrovirus-transduced cells (open circles), the GFP⁺Foxp3⁺ fraction of eMIGR1-Nr4a2 retrovirus-transduced cells (filled triangles), or the GFP⁺Foxp3⁺ fraction of eMIGR1-Nr4a2 retrovirus-transduced cells supplemented with IL-2 (5 ng ml⁻¹, crosses). Cells were cultured for 96 h with anti-CD3/anti-CD28-coated beads. The ratio of responder cells with more than three divisions are presented. Bottom: results in the top panel at the 1:1 ratio of regulatory cells/Tresp are presented. Numbers in FACS plots represent percentages of CD4⁺ T cells in the gated area. Data are representative of three independent experiments (mean and s.d. of triplicate). **P<0.01 (two-tailed Student′s t-test).