The following antibodies were used for flow cytometry and cell sorting: phycoerythrin (PE)conjugated anti-CD25 (4E3;

Miltenyi Biotec, Bergisch Gladbach, Germany), fluorescein FITCconjugated anti CD4 (13B8.2; Beckman Coulter, Fullerton, CA, U.S.A.), PE-conjugated antiCD148 (143-4; R&D systems, Minneapolis, MN, USA), PE-conjugated anti-IL17 (eBio64CAP17; e-Bioscience), PE-conjugated anti-CD278 (ISA-3, e-Bioscience), PerCP-conjugated anti-CD3 (SK7), PerCP-conjugated anti-CD4 (L200), FITC-conjugated anti-CD147 (HIM6), APCconjugated anti-CD71 (M-A712), APC-conjugated anti-CD95 (DX2), AlexaFluor 647conjugated anti-CD127 (HIL-7R-M21), Alexa 647- conjugated anti-FoxP3 (259D/C), PEconjugated anti-IFN-g (25723.11), PE-conjugated anti-TNF- (MAb11), PE-conjugated antiIL10 (JES3-9D7), PE- conjugated anti-CD152(CTLA-4) (BNI3), PE-conjugated anti-CD62L (Dreg 56), PE-conjugated CD38 (HB-7), PE-conjugated anti-CD45RA (HI100), PE-conjugated antiCD45RO (UCHL1), PE-conjugated anti-IL2 (MQ1-17H12) were all purchased from BD Bioscience (San Jose, CA U.S.A). Antibodies used for western blot analysis were purchased from Abcam; anti-FoxP3 (ab4728-100), Santa Cruz (Santa Cruz Biotechnology, Inc. Santa Cruz, CA. U.S.A.); anti-FACE1 (sc66886) and anti-MFN2 (sc50331) and Novus Biologicals (Novus Biologicals, LLC, Littleton, CO, U.S.A); anti-DJ-1 (NB300-270). Methylation analysis The 5 methylation status of the Treg-specific demethylated region (TSDR) in the FoxP3 gene was analyzed as described by Baron et al. and Miyara et al.1,2. In brief, CD4+ enriched PBMC

from a male donor were purified using Ficoll gradient centrifugation combined with RosetteSep Human CD4+ T Cell Enrichment Cocktail (STEMCELL Technologies). After staining with CD4-PerCP (BD, clone L200), CD25-PE (Miltenyi, clone 4E3) and CD147-FITC (BD, clone HIM6), CD4+CD25-CD147++, CD4+CD25++CD147++, CD4+CD25-CD147- and CD4+CD25++CD147cells were FACS sorted on a FACSAria (BD). Subsequently, DNA was extracted from the purified cell populations (DNeasy Blood & Tissue Kit, Qiagen) followed by a complete bisulfite conversion (EpiTect Bisulfite Kit, Qiagen) and PCR using Fxpro-met_F1 and Fxpromet_R2 based primers.2 PCR products were then cloned into the pGEM-T Easy vector (Promega) and sequenced using the M13 standard forward primer.

Table S1 The table lists all proteins observed exclusively in CD4+CD25-depleted T cells, number of observations and observed peptides, total MASCOT score and the proteins respective cellular compartments. Proteins were exclusively identified in CD4+CD25 depleted cells and in at least two out of three experiments. A Mascot ion score cut-off of 30 was applied on the peptide level to ensure high quality peptide mass spectra.

Figure S1 The membrane proteins Mitofusin-2 (MFN2) and CAAX prenyl protease 1 homolog (FACE1) are preferentially expressed in Tregs. FACS sorted CD4CD25highCD127low and CD4+CD25lowCD127high T cells were analyzed for their expression of mitofusin-2 (MFN2) and CAAX prenyl protease 1 homolog (FACE1) by Western blotting. FoxP3 and PARK7 were used as positive and loading control, respectively.

Figure S2 DNA methylation analysis of the 5 region of the FoxP3 gene. Four populations of CD4+ cells from a male donor were FACS sorted based on their expression of the surface markers CD25 and CD147: the CD25-CD147++ (upper left gate), the CD25++CD147++ (upper right gate), the CD25-CD147- (lower left gate) and the CD25+CD147(lower right gate) and analyzed for the methylation pattern of a 5 region in the FoxP3 gene, the Treg-specific demethylated region (TSDR). The circles indicate the average percentage of demethylation of ten CpG methylation sites (-256, -216, -139, -127, -114, -78, -66, -59, -44 and -16), that were derived from sequence analysis of several clones as described by Miyara

et al2 (CD25-CD147++: 2 clones, CD25++CD147++: 5 clones, CD25-CD147-: 4 clones, CD25++CD147-: 5 clones).

REFERENCES 1. Baron U, Floess S, Wieczorek G et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3+ conventional T cells. Eur. J. Immunol. 2007; 37: 2378-89.

2.

Miyara M, Yoshioka Y, Kitoh A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009; 30:899-911.

Table S1

List of proteins uniquely observed in CD4+CD25-depleted cells as compared to CD4+CD25-enriched T cells

Uniprot accession HYES_HUMAN NFAC2_HUMAN ADCK3_HUMAN NCOA5_HUMAN ABLM1_HUMAN GRAA_HUMAN GRAK_HUMAN NOC2L_HUMAN NOG1_HUMAN NOSIP_HUMAN NUP85_HUMAN RBM10_HUMAN REQU_HUMAN SSF1_HUMAN

Description Epoxide hydrolase 2 Nuclear factor of activated T-cells Chaperone activity of bc1 complex like Nitric oxide synthase-interacting protein Actin-binding LIM protein 1 Granzyme A Granzyme K Nucleolar complex protein 2 homolog Nuclear receptor coactivator 5 Nucleolar GTP-binding protein 1 Nucleoporin NUP85 RNA-binding protein 10 Suppressor of SWI4 1 homolog Zinc finger protein ubi-d4

no. observations 3 2 3 3 3 3 3 3 3 3 3 2 2 3

#pept. pr. exp. 5 2 2 2 3 2 1 3 2 3 3 2 1 1

Mascot score 241 83 126 118 188 71 71 89 95 103 151 91 50 97

Subcell. location Cytosol Cytosol Membrane Membrane Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos. Nucleus/ins. cytos.

Figure S1

CD

4+ CD

25 h igh CD

12 CD

7 lo

w 4+ CD

25 l o

w CD

MFN2

FOXP3

FACE1

PARK7

12

7 hi gh

Figure S2

CD4+

3.1%

0.6%

90
CD25-CD147++

94
CD25++CD147++

CD147

46.6%

1.8%

25
CD25-CD147-

100
CD25++CD147-

CD25