The antibodies used in this work are listed

in Supporting Information Table 1. DNA primers were purchased from TIB-Molbiol (Berlin, Germany) and Life Technologies (Darmstadt, Germany) and listed in Supporting Information Tables 2 and 3. EL4 cells were cultured in DMEM medium. RLM11 and primary T cells were cultured in RPMI1640 medium. Both media were supplemented with 10% FCS. BMDMs were grown as described [107]. Human CD4+ cells were isolated using magnetic-activated cell sorting (MACS) technology (Miltenyi find more Biotec, Bergisch Gladbach, Germany) from blood of healthy volunteers (DRK, Berlin, Germany), collected according to the rules of the local ethics committees on human studies (Charité, Berlin, Germany). Mouse total CD4+ T and naive CD4+CD25−CD62L+ cells were isolated from spleen, mesenterial, popliteal, and auxiliary lymph nodes by MACS. CD4+ T cells from FoxP3-IRES-GFP mice were fractionated into FoxP3+ and FoxP3− cells by fluorescence-activated cell sorting (FACS) technology using FACSAria or FACSDiVa flow cytometers (BD Biosciences, Franklin Lakes, NJ, USA). Naive T cells were mixed with irradiated CD4− cells at the ratio of 1:5 and polarized under CHIR 99021 Th1, Th2, and Th17 conditions (summarized

in Supporting Information Table 4). Polarization efficiency was assessed by measurement of lineage-specific cytokines (Supporting Information Fig. 10). Restriction enzyme accessibility assay was performed as described [108]. All enzymes were from New England Biolabs (Ipswich, MA, USA). Briefly, cells were washed with ice-cold PBS, centrifuged for 5 min at 500 × g, resuspended in lysis buffer 1 Quisqualic acid (L1) (10 mM TrisHCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 0.15 mM spermine, and 0.5 mM spermidine) and incubated on ice for 5 min. Nuclei were centrifuged for 5 min at 500 × g, washed and resuspended in 50 μL of appropriate restriction enzyme buffer. A total of 30 U of restriction enzyme were added, and nuclei were incubated at 37°C for 15 min. The reaction was stopped

by adding 450 μL of DNA isolation buffer (100 mM NaCl, 10 mM TrisHCl, pH 8.0, 25 mM EDTA, 0.5% SDS), supplemented with 10 μL of 20 mg/mL Proteinase K (Biodeal, Markkleeberg, Germany) and incubated for 2 h at 56°C with shaking. Then, 300 μL of 3 M NaCl were added, samples were vortexed, and centrifuged for 15 min at 20 000 × g and 4°C. Supernatants were transferred to new tubes, supplemented with 10 μg of glycogen, and mixed with 750 μL of isopropanol. DNA was precipitated by 30 min centrifugation at 20 000 × g and 4°C, washed with 70% ethanol, dried, resuspended in 5 mM TrisHCl, pH 8.5, and analyzed by Southern blotting. Cells were fixed for 10 min with 1% formaldehyde in PBS at room temperature (RT). The fixation was stopped by adding glycine to the final concentration of 125 mM, cells were incubated for 5 min at RT, washed with cold PBS, resuspended in L1 buffer, and incubated for 10 min on ice.