Antibodies, Cytokines and Chemicals

Supplemental Information:

Antibodies, cytokines and chemicals:

Rat anti-mouse CD4 mAb (L3T4), rat anti-mouse FoxP3 mAb (FJK-16a) both eBioscience; hamster anti-mouse CD95 mAb (Jo2) from BD; 7AAD (Sigma-Aldrich); mouse anti-mouse Ly5.1 mAb (A20), mouse anti-mouse Ly5.2 (104), rat anti-mouse CD25 mAb (3C7), rat anti-mouse CD45RB mAb (C363-16A), hamster anti-mouse CTLA-4 mAb (UC10-4B9), rat anti-mouse IL-4 mAb (11B11), rat anti-mouse IFN- (XMG1.2), rat anti-mouse IL-17 mAb (TC11-18H10.1) and AnnexinV-Alexa647, all Biolegend. For intracellular staining of cytokines and Foxp3 cells were fixed and permeabilized following to the manufacturer’s instructions (Biolegend). For quantification of mitotic cells, ethanol-fixed cells (70% EtOH in distilled water) were permeabilized with 0.25% Triton-X-100 in 0.1% sodium citrate, followed by staining with a rabbit-anti-phospho-H3 (Ser10)-specific antibody (Cell Signalling) and donkey anti-rabbit Alexa-647 antibody (Jackson Immuno Research). RNA was digested with 10µg/ml RNase A (Sigma-Aldrich) in PBS and DNA stained with 1 µg/ml 7AAD. Cytokines and/or inhibitors were used in the following concentrations: 100U/ml IL-2, 5ng/ml TGF-, 100U/ml IL-2 + 5ng/ml TGF-, 100nM Rapamycin (Alexis) + 100U/ml IL-2, 10µM LY294002 (InvivoGen)± 100 U/ml IL-2, 100ng/ml Cyclosporin-A (LC Laboratories)± 100 U/ml IL-2, 5 µM U0126 (Cell Signaling) ± 100 U/ml IL-2.

Primers used for qPCR analysis

qPCR was performed using the following primers (5´-3´): Actin, ACTGGGACGACATGGAGAAG (sense) and GGGGTGTTGAAGGTCTCAAA (antisense); Bcl-2, CTGGCATCTTCTCCTTCCAG (sense) and GACGGTAGCGACGAGAGAAG (antisense); Bcl-xL TTCGGGATGGAGTAAACTGG (sense) and TGGATCCAAGGCTCTAGGTG (antisense); Bid, CTGCCTGTGCAAGCTTACTG (sense) and GTCTGGCAATGTTGTGGATG (antisense); Bim; GAGATACGGATTGCACAGGA (sense) and TCAGCCTCGCGGTAATCATT (antisense), Bmf, CCCATAAGCCAGGAAGACAA (sense) and AGGGAGAGGAAGCCTGTAGC (antisense); CD122 GAAGTGCTCGACGGAGATTC (sense) and GAAGTAGCCCTGGTTGGTGA (antisense); CD95L, ACTCCGTGAGTTCACCAACC (sense) and GTGGGGGTTCCCTGTTAAAT (antisense); cFLIP, aaccctcacctggtttctga (sense) and ccttggctatcttgcctctg(antisense); (GATA-3, CTGGAGGAGGAACGCTAATG (sense) and TCTGGATGCCTTCTTTCTTCA (antisense); IL-2, AACCTGAAACTCCCCAGGAT (sense) and CGCAGAGGTCCAAGTTCATC (antisense); p21, TTGCACTCTGGTGTCTGAGC (sense) and TCTGCGCTTGGAGTGATAGA (antisense); RORt, CGACTGGAGGACCTTCTACG (sense) and TTGGCAAACTCCACCACATA (antisense); T-bet, GGTGTCTGGGAAGCTGAGAG (sense) and GAAGGACAGGAATGGGAACA (antisense).

MethyLight PCR primers

Three Foxp3 assays (two reactions for DNA methylation analysis and one for internal reference) were determined with the assistance of the computer program Primer Express version 2.0.0 (Applied Biosystems, Foster City, CA, USA) to produce a 157-base-pair, a 115-base-pair and a 131-base-pair PCR amplicon (Foxp3_01: nucleotide positions 752.060 – 750.589, Foxp3_02: nucleotide positions 757.960 – 758.074 and Foxp3 Reference: nucleotide position 750.433 – 750.563 as defined by NCBI Reference Sequence NT_039700.7|MmX_39740_37) with a mean distance of -2.226 base pairs, +3.653 base pairs or -3.866 base pairs respectively to the transcription start site.The following primers (5’ to 3’ direction) were used for MethyLight PCR: Foxp3_01: Forward: CAACCTAAACTTAACCAAATTTTTCTACC, Reverse: GGGTTTATTCGGTTATAGGATAGATTAGTTATT, TQM-probe: 6-FAM-ACGTCATAACGACCGAATACATTAAACTTCATCGA-BHQ1; Foxp3_02: Forward: CCACCTACCTCTACCTCCCAAATA, Reverse: AAATTTGAGAGGGAAAA GTGATTGTT, TQM-Probe: 6-FAM-AAACATACGCCGCCGCCACCA-BHQ1; Foxp3 Reference: Forward: GGAAAAGTGATAGAGAAAGAATGAATAAGA, Reverse: AACATCAATCCTCCA ACCAAAAA, TQM: 6-FAM-TGAGAGAAAGGATTGAAAAT-MGB-nonfluorescent quencher. The primer specificity was verified on a 100% hypermethylated mouse DNA (Zymo Research, Orange, CA, USA) that was bisulfite-modified and on an unmodified genomic DNA.