(a–e) Naïve CD4⁺Foxp3⁻ CD62L⁺ T cells from Foxp3-GFP mice activated with or without cytokines in the presence of irradiated syngenic APCs with 1 μg/ml anti-CD3. (a) Flow cytometry for GFP expression in CD4⁺ T cells 72h after activation in the presence of TGF-β, IL-4 or both. Data represent one of three experiments. (b) ELISA assay or bead array for the indicated cytokines in 48 h culture supernatants. Data represent one of two experiments. (c) Flow cytometry for intracellular expression of IL-10, IL-4, IL-17 and IFN-γ in CD4⁺ T cells measured at day 4 post-activation. Data represent one of three experiments. (d) Proliferation of cytokine-treated WT T cells was measured by ³H-thymidine incorporation in triplicate wells of T cells activated in the presence of irradiated syngenic APCs with 1 μg/ml anti-CD3. Proliferation was assessed in triplicate wells and is presented as mean ± s.d. Data represent one of three experiments. (e) Quantitative RT-PCR for the indicated transcripts for various transcription factors (mRNA expression relative to Gapdh). Data represent mean one of two experiments.

(a) RT-PCR for Foxp3 mRNA induction in IL-4 transgenic T cells (IL-4 Tg) and wild-type T cells (WT) after treatment by anti-CD3 + anti-CD28 and the indicated doses of TGF-β. Data represent one of 2 experiments. (b–d) Naïve CD4⁺Foxp3⁻CD62L⁺ T cells from STAT6KO mice activated with or without cytokines in the presence of irradiated syngenic APCs and 1 μg/ml of anti-CD3. Flow cytometry for Foxp3-EGFP expression at 72 h (b), for intracellular Foxp3-EGFP and IL-10 expression at day 4 (c), and RT-PCR for IL-9 mRNA relative expression at day 4 (left) and ELISA for IL-9 protein production at day 2 (right). Data represent one of two experiments (d). (e) RT-PCR for IL-9 (left) and IL-10 (right) mRNA expression in naïve CD4⁺CD62L⁺ T cells from GATA-3-deficient.cre⁺(GATA-3KO) mice activated with or without cytokines in the presence of irradiated syngenic APCs and 1 μg/ml of anti-CD3; expression was analyzed relative to HPRT. ELISA assay for IL-9 production (middle) was also evaluated. (f) Suppression assay with naïve STAT6KO responder cells cultured with T_reg cells purified from either wild-type (WT) or STAT6KO mice in presence of the indicated cytokines (left). Proliferation was assessed in triplicate wells and is presented as mean ± s.d. Data represent one of two experiments. Percent suppression in each culture was calculated relative to the proliferation of effector T cells alone (T_eff) (right).

(a) RT-PCR for Foxp3 and GATA-3 expression in CD4⁺T cells from Gata3^flox/flox mice transduced with a retroviral vector expressing Cre; the cells were also treated with TGF-β (3 ng/ml), as indicated, to induce Foxp3 expression. RNA was extracted from the cells 48 hr later and expression of mRNAs was measured relative to HPRT. Data represent one of two experiments. (b) Luciferase assay on HEK-293 cells transfected with a Foxp3-renilla construct and plasmids expressing the indicated Flag-tagged GATA-3 or STAT6 or a control vector (pCDNA). Immunoprecipitation was performed using anti-FLAG coated beads and then the interaction of Foxp3 with GATA-3 and/or STAT6 was determined by monitoring the luciferase activity. Data are representative of three experiments. (c) RT-PCR for expression of the indicate mRNAs in CD4⁺ T cells activated under T_H2-polarizing condition and transduced with either a control retroviral vector or Foxp3-GFP-expressing vector. Foxp3-GFP⁺ cells were sorted 48 hours later and expression of indicated cytokine transcripts was analyzed relative to HPRT. Alternatively, the Foxp3-GFP-transduced T cells were cultured for 3 more days in presence of IL-2 and then re-stimulated for 48 hours using plate bound anti-CD3 and anti-CD28. Supernatants were collected and cytokine analysis was performed by ELISA (right graph). Data presented are representative of three experiments. (d) Luciferase assay on Jurkat cells transfected with an Il5 promoter-luciferase construct and plasmids expressing Foxp3, GATA-3 or both, as indicated for 24 hours. The cells were then activated for 3–4 hours with PMA + ionomycin and luciferase activity was measured. Data presented are representative of three experiments.

(a) Suppression assay with naïve CD4⁺CD62L⁺ T cells from IL-10.GFP reporter mice activated with or without cytokines (IL-4 + TGF-β) and non-treated IL-10.GFP⁺ T cells. Naïve wild-type Foxp3⁻ responder T cells were cultured alone, together with natural-occurring T_regs or with IL-10.GFP⁺ cells stimulated with TGF-β plus IL-4 (T4-IL-10.GFP⁺ cells); when indicated, anti-IL-9 or anti-IL-10 was added to the culture. Proliferation was assessed in triplicate wells by ³H-thymidine incorporation and is presented as mean ± s.d. Data represent one of three experiments. (b) RAG-1-deficient mice were reconstituted with sorted CD4⁺Foxp3-GFP⁻CD45RB^hiT cell sub populations in PBS. Mice received CD4⁺Foxp3-GFP⁻CD45RB^hiT cell alone or in combination with IL-10.GFP⁺ cells stimulated with TGF-β plus IL-4 (T4-IL-10.GFP⁺ cells) or CD4⁺Foxp3⁺ naturally occurring T_reg cells. Mice were examined for their weight loss for 9 weeks. Each time point represents an average of the calculated weight of all mice present per group (5–8 mice) of 2 independent experiments. The total colonic score was calculated as the average of the individual scores from the sections of proximal colon, mid-colon, and distal colon28. The mice in Group 1 (CD45RB^hi effector T) had a pathology score of 7.5 ± 3.5; Group 2 (CD45RB^hi effector T cells + (TGF-β + IL-4) IL-10.GFP⁺ cells): 8.8±2.3; Group 3, (T4-IL-10.GFP⁺ cells): 5.7±2.6 and Group 4, (CD45RB^hi T_eff cells + T_reg cells) : 3.4±1.5. The difference in the mean score was statistically different between Group 2 and 4 (P = 0.01) and Group 3 and 4 (P = 0.03) (Mann-Whitney one tailed U test). (c) Representative histopathology of colitis and peripheral neuritis in Rag1^−/−mice reconstituted with the following cell types. Group 1, CD45RB^hi effector T cells (mononuclear cell inflammation in the lamina propria, muscularis and adventitial tissues (arrow)). Group 2, CD45RB^hi effector T cells + (TGF-β + IL-4) IL-10.GFP⁺ cells (T4-IL-10.GFP⁺ cells); inflammation in the lamina propria and submuscularis (arrow) and blue staining of myelin; higher power of a nerve root (middle) with inflammatory cell infiltration (arrows) and myelin breakdown product in vacuoles (right, arrow). Group 3, T4-IL-10.GFP⁺ cells alone; inflammation in the lamina propria, muscularis mucosa and adventitial tissues (left, arrow); a spinal nerve root shows vacuolation with myelin breakdown products (right, arrows). Group 4, CD45RB^hi T_eff cells + T_reg cells; intact mucosal architecture and the muscularis (left), intact spinal cord white matter and nerve root (middle) and higher power of intact myelin in cauda equina nerve root (right) a–d. Luxol fast blue-H&E, e–h. Bar in Group 1 corresponds to 200 μm, in 100 μm in c and h. Bar in D corresponds to 100 μm in D, F and H.