Anti phycoerythrin microbeads

Naive CD4⁺ T cells (CD62L⁺CD44⁻ or CD62L⁺CD44⁻FoxP3GFP⁻) were sorted from WT B6, Irf4^−/−, or Foxp3gfp reporter mice by a FACSAria flow cytometer. B6 APCs were prepared by depletion of T cells from B6 splenocytes with phycoerythrin–anti-CD3 (clone 2C11; BioLegend) and anti-phycoerythrin microbeads (Miltenyi Biotec, San Diego, CA), followed by brief treatment with 50 μg/ml mitomycin C (Fisher Scientific) before each experiment. For activation of T cells, naïve CD4⁺ T cells were added at 1×10⁵ cells/well in 96-well round bottom tissue-culture plates (Thermo Fisher Scientific), and stimulated with equal numbers of B6 APCs and 1 μg/ml soluble anti-CD3e mAb (clone 2C11; BioLegend). For some experiments cell cultures were supplemented with various cytokines (PeproTech, Rocky Hill, NJ) and small-molecule inhibitors (Selleck Chemicals). In some cases, naïve CD4⁺ T cells were labeled with CellTrace™ Violet reagent prior to stimulation. CD4⁺ T cells cultured for different days were collected and analyzed with flow cytometry, microarray analysis, Immunoblot, and quantitative real-time PCR, and ChIP.

Single-cell suspensions were prepared from the spleen and liver. Hepatic iNKT cells were isolated from the liver as described previously using 34% Percoll solution (Sigma, St Louis, MO, USA). The following antibodies were used: TCRβ FITC (clone H57-597), CD4 FITC (L3T4), CD1d tetramer Alexa 488 (NIH Tetramer Core Facility (mCD1d/PBS57)), CD1d tetramer PE (NIH Tetramer Core Facility (mCD1d/PBS57)), TCRβ PE (H57-597), PLZF PE (Mags.21F7), NK1.1 PerCP Cy5.5(PK136), NK1.1 APC (PK136), TCRβ APC (H57-597), TBET APC (4B10), CD1d tetramer Alexa 647 (NIH Tetramer Core Facility (mCD1d/PBS57)), CD19 PeCy7(ID3) and Ki-67 Pacific Blue (SolA15). All antibodies were purchased from eBioscience (San Diego, CA, USA), unless otherwise specified. Samples were collected on a FACS (fluorescence-activated cell sorting) LSRII, FACS Fortessa or FACS Aria (BD Biosciences, San Jose, CA, USA) fitted with custom mirrors from Omega Filters (510/21) with 502LP or 505LP for GFP, 530/30 with 525LP for YFP) and were analyzed with FlowJo software (TreeStar, Ashland, OR, USA). For iNKT cell sorting, single-cell suspensions were MACS-enriched for CD1d tetramer phycoerythrin-stained cells using magnetically labeled anti-phycoerythrin microbeads (Miltenyi Biotec, Auburn, CA, USA). CD19⁻CD3⁺CD1d tetramer⁺ iNKT were then sorted with an FACSAria (BD Biosciences) to at least 98% purity.