Generation and Characterization of Conditional Foxp3 Mutant Mice

CNS1KO (Foxp3^ΔCNS1), Foxp3^GFP, Foxp3^Thy1.1 and Foxp3^DTR mice were previously described^{7 (link), 22 (link), 23 (link)}. Male C57BL/6 (B6) mice were purchased from The Jackson Laboratory. and groups of 5 co-housed mice were randomly assigned to treatment vs. control groups after confirmation that age and weight were in accordance between groups. Male mice were used for all experiments. All strains were maintained in the Sloan-Kettering Institute animal facility in accordance with institutional guidelines. For antibiotic treatment, mice were given 1 g L⁻¹ metronidazole (Sigma-Aldrich), 0.5 g L⁻¹ vancomycin (Hospira), 1 g L⁻¹ ampicillin (Sigma-Aldrich) and 1 g L⁻¹ kanamycin (Fisher Scientific) in drinking water (AVNM). For butyrate, acetate and propionate administration, each SCFA was added to AVNM-containing drinking water at 36 mM and pH-adjusted as needed. DCs were expanded in vivo by subcutaneous injection of B16 melanoma cells secreting FLT3-ligand and purified using CD11c (N418) magnetic beads (Dynabeads, Invitrogen). In vitro Foxp3 induction assays were performed by incubating 5.5 × 10⁴ FACS-sorted naïve CD44^loCD62L^hiCD25^-CD4⁺T cells with 1 μg ml⁻¹ of CD3 antibodyin the presence of DCsin 96-well flat-bottom plates for 4 d. Alternatively, naïve CD4⁺T cells were stimulated with CD3 and CD28 antibody-coated beads (Dynabeads Mouse T-Activator, Invitrogen) at a 1:1 cell-to-bead ratio. All cultures were supplemented with 1 ng mL^-1 TGF-β and 100 U ml⁻¹ IL-2. Intracellular staining for IL-17, IFN-γ, IL-4, IL-13 and Foxp3 was performed using the Foxp3 staining kit (eBiosciences). Cytokine staining was performed after re-stimulation of ex vivo isolated cells with 5 μg ml⁻¹ CD3 antibody and 5 μg ml⁻¹ CD28 antibody in the presence of Golgi-plug (BD Biosciences) for 5 h. Stool samples were collected directly into sterile tubes from live mice and snap-frozen before preparation of material for SCFA quantification by HPLC or LC-MS. HPLC analysis of 2-nitrophenylhydrazine HCl-derivatized SCFA present in stool extracts was performed as describedelsewhere ^{24 (link)}. H3K27Ac ChIP-qPCR was performed as previously described^{25 (link)}.