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
10 µg/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: 100 U/ml IL-2,
5 ng/ml TGF-, 100 U/ml IL-2 + 5 ng/ml TGF-, 100 nM Rapamycin (Alexis) + 100
U/ml IL-2, 10 µM LY294002 (InvivoGen) ± 100 U/ml IL-2, 100 ng/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
ACTGGGACGACATGGAGAAG
(antisense);
Bcl-2,
the
(sense)
following
and
primers
(5´-3´):
Actin,
GGGGTGTTGAAGGTCTCAAA
CTGGCATCTTCTCCTTCCAG
(sense)
and
GACGGTAGCGACGAGAGAAG (antisense); Bcl-xL TTCGGGATGGAGTAAACTGG
(sense)
and
TGGATCCAAGGCTCTAGGTG
CTGCCTGTGCAAGCTTACTG
(antisense);
Bim;
(sense)
and
(antisense);
Bid,
GTCTGGCAATGTTGTGGATG
GAGATACGGATTGCACAGGA
(sense)
and
TCAGCCTCGCGGTAATCATT (antisense), Bmf, CCCATAAGCCAGGAAGACAA
(sense)
and
AGGGAGAGGAAGCCTGTAGC
GAAGTGCTCGACGGAGATTC
(antisense);
CD95L,
(sense)
and
(antisense);
CD122
GAAGTAGCCCTGGTTGGTGA
ACTCCGTGAGTTCACCAACC
(sense)
and
GTGGGGGTTCCCTGTTAAAT (antisense); cFLIP, AACCCTCACCTGGTTTCTGA
(sense)
and
CCTTGGCTATCTTGCCTCTG
CTGGAGGAGGAACGCTAATG
(antisense);
IL-2,
(sense)
and
(antisense);
(GATA-3,
TCTGGATGCCTTCTTTCTTCA
AACCTGAAACTCCCCAGGAT
(sense)
and
CGCAGAGGTCCAAGTTCATC (antisense); p21, TTGCACTCTGGTGTCTGAGC
(sense)
and
TCTGCGCTTGGAGTGATAGA
CGACTGGAGGACCTTCTACG
(antisense);
T-bet,
(sense)
and
(antisense);
RORt,
TTGGCAAACTCCACCACATA
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 157base-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-FAMACGTCATAACGACCGAATACATTAAACTTCATCGA-BHQ1; Foxp3_02: Forward:
CCACCTACCTCTACCTCCCAAATA,
Reverse:
AAATTTGAGAGGGAAAA
GTGATTGTT, TQM-Probe: 6-FAM-AAACATACGCCGCCGCCACCA-BHQ1; Foxp3
Reference:
Forward:
GGAAAAGTGATAGAGAAAGAATGAATAAGA,
Reverse:
AACATCAATCCTCCA ACCAAAAA, TQM: 6-FAM-TGAGAGAAAGGATTGAAAATMGB-nonfluorescent quencher. The primer specificity was verified on a 100%
hypermethylated mouse DNA (Zymo Research, Orange, CA, USA) that was bisulfitemodified and on an unmodified genomic DNA.