text: Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation
  To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells. 
  