Figure 4

SMAD3 mediated transcriptional regulation of FOXP3 in CD8⁺ Treg cells. (A) Multiple sequence alignment between human FOXP3 and mouse Foxp3 depicted the non-coding conserved sequence; viz. CNS1, CNS2, CNS3. Criteria that were applied for rVista analysis required 80% identity for at least 100-bp length. (B) Lymphocytes were isolated from a healthy individual and were cultured in media alone or in in vitro-generated tumor milieu in presence or absence of TGFβ receptor inhibitor SB431542 and a percent population of CD8⁺CD25⁺FOXP3⁺ Treg cells were analyzed by flow cytometry (left panel). The graphical representation depicted percent CD8⁺CD25⁺FOXP3⁺ Treg cells generated in SB431542-treated/TGFβ-neutralizing antibody-treated conditions (right panel). (C) Graphical plot represents the relative changes in phospho-SMAD3 levels in the SB431542-treated control CD8⁺ cell as well as CD8⁺FOXP3⁺ Treg cells. (D) Representative confocal images depicted nuclear translocation of p-SMAD3 in control CD8⁺ cells and tumor-CD8⁺ Treg cells (magnification 60x; scale bar: 5 μM) (left panel). Nuclear translocation of p-SMAD3 was analyzed by ImageJ software and depicted graphically (right panel). (E) The relative change in FOXP3 expression at transcript and protein level was determined in control CD8⁺ cell and SMAD3-siRNA-transfected CD8⁺ Treg cells by qPCR and flow cytometry respectively. Transfection efficiency was determined by Western blot (inset). (F) rVista alignment of human/mouse/guinea-pig FOXP3 genome sequences. Criteria required 90% similarity for at least 35 bp length. The conserved regions were in the red. The outlined area indicated SMAD3-binding sites in CNS1 enhancer region (above). Sequence conservation of the SMAD3-binding site in CNS1 region of FOXP3 genome between human/mouse/guinea-pig was analyzed by CLUSTALW (bottom). The highlighted area indicated SMAD3-binding sequence. (G) Schematic representation of SMAD3-binding sites at CNS1 region and the used primer set: +1392 bp to +1599 bp (above). In the bottom, qPCR data showed relative SMAD3 binding (left panel) and histone-H4 acetylation (right panel) in control CD8⁺ T cells and control-/SMAD3-siRNA transfected tumor-CD8⁺ Treg cell. Data are representative as the mean ±SEM and are the cumulative results of five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.