We thank J. anti-CD3 (17A2) and 1g/ml of soluble anti-CD28 (37.51). Cells were then stimulated with indicated cytokines (10ng/ml of IL-1, 20ng/ml of IL-6, 10ng/ml of IL-12, 10ng/ml of IL-23, 10nM of RA, 2ng/ml of TGF- (Treg), 0.2ng/ml of TGF- TH17) in RPMI (Invitrogen) containing 10% FCS (Sigma), 1% L-glutamine (Gibco), 25mM HEPES (Gibco), 1% essential amino acid mixture (Gibco), 5M -mercapto ethanol and 1% pen-strep antibiotics (Gibco). Where indicated, cells were stimulated in serum-free media X-VIVO 20 (Lonza) supplemented with the after mentioned components. For block of leptin signaling, cells were incubated with 250ng/ml of mouse leptin receptor fusioned to Fc portion of immunoglobulin (LepR:Fc chimera (R&D)). For re-stimulation experiments, cells were cultured for 4.5 days as above and resuspended in new media containing the indicated cytokines for another 72h. Quantitative PCR (q)PCR was performed as previously described (11). RPL32 housekeeping gene was used to normalize samples. Primers used: contamination Mice were infected with 2 108 of per animal, as previously described (12). Bacteria were inoculated by gavage in recipient mice in a total volume of 200l of sterile PBS. After contamination, mice were followed daily for weight loss and colony forming units (CFU) in feces and liver. Mice were sacrificed and analyzed 18 days after contamination. Leptin activity by imaging imaging of transgenic animals were performed using the Xenogen IVIS Lumina imaging system (Caliper). Anesthetised animals were injected intraperitonally with luciferin (200 l of stock 15 mg/ml in PBS). After 15 to 20 min, the animals were imaged in an imaging chamber and the photon image was analyzed by Living Image 3.0 software (Xenogen). Phosphorylated and total STAT3 Western blot analysis Na?ve (defined as CD4+CD25?CD62hiCD44lo) T cells were sorted using Amicarbazone FACS Aria cell sorter flow cytometer (Becton Dickinson) and rested for 30 minutes at 37C in serum free medium. Cells were then stimulated with 20ng of IL-6 for 30 minutes and protein was extracted at 4C for 15 minutes using RIPA buffer plus Phospho Stop (Roche 04-906-837-001) and proteinase inihibitor (Calbiochem 539-134). Cell protein extract was subjected to eletrophoresis separation and transfer to PVDF membrane. The membrane was blocked for 1 hour with TBS-T 5% milk, incubated overnight with anti-phospho-STAT3 antibody (Cell Signaling Y705) and developed using secondary antibody conjugated to HRP. Anti-total STAT3 antibody (Cell Signaling 79D7) was used as a control. Induction of Experimental Alergic Encephalomyelitis (EAE) Female animals were immunized with 100g of MOG peptide emulsified in CFA 1:1 mixture intradermic in the flank. Animals were inoculated 4 hour before and 2 days after immmunization with 200ng of pertussis toxin (Sigma). Animals were Amicarbazone monitored Amicarbazone daily for weight loss and EAE symptoms. Animals were scored according to an established scoring system: level 1, Rabbit Polyclonal to GATA2 (phospho-Ser401) limp tail; level 2, hind leg weakness or partial paralysis; level 3, total hind leg paralysis; level 4, hind leg paralysis and front leg weakness or partial paralysis; level 5, moribund. Preparation of intraepithelial and lamina propria lymphocytes Intraepithelial and lamina propria lymphocytes were isolated as previously described (11). Statistics Statistical analyses were performed in GraphPad Prism software. Data was analyzed by applying one-way ANOVA or unpaired Students value of less than 0.05 was considered significant. RESULTS Leptin receptor signaling is required for Th17 differentiation To study the cell-intrinsic role of leptin signaling on T cells, we generated CD4-driven enhancer/promoter, which avoids targeting of other CD4+ cell populations such as innate lymphoid cells (ILCs) (13). PCR analysis for floxed exon 1 of.