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. 2007 Oct 31;104(46):18169–18174. doi: 10.1073/pnas.0703642104

Fig. 4.

Fig. 4.

Negative cross-regulation of Foxp3+ Treg development by Th1/Th2 effector cytokines and the lineage-specific transcription factors. (A) Naïve CD4+ cells from WT mice were stimulated in primary cultures (1° stimulation) by anti-CD3/anti-CD28 under different priming conditions: 1, nonpolarizing, no neutralizing mAbs or Th1/Th2-polarizing cytokines; 2, Th1-polarizing plus 10 ng/ml IL-12; 3, Th2-polarizing plus 10 ng/ml IL-4; 4, neutral plus neutralizing mAbs (10 μg/ml) to IFNγ and IL-4. The cells were harvested on day 7 and restimulated by plate-bound anti-CD3 and TGF-β1 in secondary cultures (2° stimulation) with or without neutralizing mAbs (10 μg/ml) to IFNγ and/or IL-4 to induce Foxp3+ Treg. A rat IgG isotype control was included (control Ab). Representative FACS plots of Foxp3 staining are shown (average of two independent experiments). (B) The cells from A were restimulated in the presence of anti-IFNγ/IL-4 to induce Foxp3+ Treg differentiation. Suppressive activities of the induced Tregs were determined by CFSE-based suppression assay (average of two independent experiments). Naïve CD4+ cells from WT (C), IFNγ−/− (D), or IL-4−/− (E) mice were activated under the neutral condition and transduced with an EGFP bicistronic retroviral vector encoding T-bet or GATA-3. Empty vector was included as control (Vector ctrl). The transduced cells (GFP+) were isolated by cell sorting for subsequent experiments. (Top and Middle) Intracellular cytokine staining for IFNγ and IL-4 production. (Bottom) Foxp3 expression induced by anti-CD3 plus TGF-β1 stimulation in secondary culture (average of two independent experiments). (F) The cells from C–E were subjected to real-time PCR analysis for Foxp3 mRNA expression, and relative expression levels are plotted. (G) WT CD4+ T cells were transduced with the mutant variants of T-bet and GATA-3, and Foxp3 induction was as in C. Histograms of Foxp3 expression are shown. T-bet ΔDBD, deletion of the T-box DNA binding domain; T-bet ΔAD, deletion of the C-terminal transcription activation domain; GATA-3 ΔNF, deletion of the N-terminal zinc finger DNA binding domain; GATA-3 ΔND, deletion of the N-terminal activation domain.