Rorγt

Cell suspensions from spleen, mesenteric lymph nodes (MLN) and the lamina propria were prepared as described previously59 (link) and first incubated with anti-CD16/CD32 (eBioscience) to prevent nonspecific binding. Single cell suspensions were stained with antibodies against CD4, CD11b, CD25, GR1, TCR-β, CD45.1, CD45.2, IL-4, IL-13, IL-17A, IL-22, IFN-γ, EOMES, RORγt, Foxp3, (all from eBioscience), CD45RB, IL-17F and T-bet (all from Biolegend). Intracellular staining was performed as follows: cells were restimulated for 4 h with 0.1 μg ml⁻¹ phorbol 12-myristate 13-acetate (PMA) and 1 μg ml⁻¹ ionomycin (both Sigma Aldrich) plus Brefeldin A (eBioscience), washed and incubated with anti-CD16/CD32. Cells were washed and stained for surface markers indicated above and fixed in eBioscience Fix/Perm buffer, followed by permeabilization in eBioscience permeabilization buffer for 1 h in the presence of antibodies. Cells were acquired with a BD LSR2 and analysis was performed with FlowJo (Tree Star) software

Skin tissues were homogenized in tissue protein extraction reagent or mammalian protein extraction reagent (Thermo, Waltham, MA, USA). Tissue homogenates or cell lysates (20 μg of total protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against p-STAT1 (Y701), STAT1, p-STAT2 (Y690), STAT2, p-STAT3 (Y705), STAT3, p-STAT4 (Y693), STAT4, p-STAT5 (Y694), STAT5, p-STAT6 (Y641), STAT6, p-PKM2 (Y105), PKM2, Ac-lysine, p-JAK2 (Y1007/1008), JAK2, SOCS1, SOCS3, cyclin D1 (Cell Signaling Technology, Beverly, MA, USA), RORγt, c-myc (eBioscience), Sirt2, Lamin B (Santa Cruz Biotechnology, Dallas, TX, USA) and HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA).

All cell experiments were strictly prepared on ice, unless otherwise stated in other specific procedures. Cells (1×10⁶ cells/sample) were washed with fluorescence-activated cell sorting staining buffer (phosphate-buffered saline, 2% fetal bovine serum or 1% bovine serum albumin, 0.1% sodium azide). All samples were incubated with anti-Fc receptor Ab (clone 2.4G2, BD Biosciences, San Jose, CA), prior to incubation with other Abs diluted in fluorescence-activated cell sorting buffer supplemented with 2% anti-Fc receptor Ab. For intracellular cytokine staining, cells were collected and fixed for 50 min with 1mL fixation buffer (IC Fixation and Permeabilization kit, eBioscience, San Diego, CA). After washing, the fixed cells were stained. The samples were filtered immediately before analysis or cell sorting to remove any clumps. The following antibodies were used: fluorescence-conjugated anti-mouse B220 (eBioscience, RA3-6B2), CD3 (eBioscience, 145-2C11), CD4 (eBioscience, GK1.5), CD8 (Biolegend, 53-6.7), RORγt (eBioscience, B2D), IL-17A (eBioscience, eBio17B7), CD24 (eBioscience, M1/69), CD138 (Biolegend, 281-2), and IgG (Santa Cruz Biotech, F1306) antibodies. Data collection and analyses were performed on a FACS Calibur flow cytometer using CellQuest software.

Cell surface staining was performed according to standard procedures using antibodies against murine CD3, CD8, CD4, CD19, CD44 and Thy1.1 (CD90.1), all purchased from BD Pharmingen (San Diego, CA). For intracellular staining, monoclonal antibodies against IFN-γ (BD Pharmingen), IL-17A, IL-17F, MIP-1α, RORγt and T-bet (eBioscience, San Diego, CA) were used. For intracellular staining of in vitro cultures, cells were restimulated with phorbol-12-myristate 13-acetate (PMA, 10 ng/ml) and ionomycin (500 ng/ml) for 3 hours. For intracellular staining of spleen cells post-infection, cells were restimulated ex vivo with A20-TS for 6 hours. All intracellular stained cells were cultured with monensin (GolgiPlug, 1 μl/ml) and brefeldin A (GolgiStop, 0.67 μl/ml) for the last 3 hours of stimulation at 37°C (both from BD Pharmingen). After surface staining, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before intracellular staining, according to manufacturer’s instructions. Cells were analyzed with a BD LSR II flow cytometer and FlowJo v7 software (Tree Star, Inc.).

Joint draining LN cells from 6 week old KRN.g7, IDO1 ko KRN.g7, IDO2 ko KRN.g7, and dko KRN.g7 mice were harvested and stained for CD4⁺ T cells (BioLegend) and the following markers to distinguish T_H subsets: bcl6 (BD Bioscience), foxP3 (Biolegend), gata3, rorγt, T-bet (all from eBioscience) as described (1 (link)). The samples were acquired on a BD FACSCanto II flow cytometer using FACSDiva Software and analyzed using FlowJo Software.