supporting information - pnas · elisa. cells (1 × 106/ml) were cultivated as indicated for 48 h....

Supporting InformationVaeth et al. 10.1073/pnas.1203870109SI Materials and MethodsMice and Cells.Nfat2fl/fl (nuclear factor of activated T-cells) animalswere generated in A. Rao’s laboratory (Harvard Medical School,Boston, MA). Nfat1−/− × Nfat4−/−, B6-Tg (Cd4-cre) 1Cwi/Cwilbcm (European mouse mutant archive, Rome, Italy) andFoxp3-IRES-cre have been described previously (1–3). Animalswere used at 6–16 wk and maintained in accordance with in-stitutional guidelines for animal welfare. Blood from healthydonors was obtained after informed consent, in accordancewith the Declaration of Helsinki, under a protocol that receivedapproval from the Institutional Review Board from the JohannesGutenberg university hospital in Mainz.

Antibodies, Reagents, and Media. mAbs against CD3ε (145-2C11),CD28 (37.51; both BD Pharmingen), and CD4 (YTS177.9; Bio-Xcell), as well as superagonistic CD28 mAb D665 (CD28SA;Serotec), were used as previously described (4). RecombinanthIL-2 (50 U/mL), hIL-6 (50 ng/mL), mIL-12 (10 ng/mL), hIL-21(65 ng/mL), mIFN-γ (50 ng/mL), hTGF-β1 (1–5 ng/mL), andmIL10 (20 ng/mL; all PeproTech), anti-IL-2 (5 μg/mL; eBio-science), anti-IL-4 and anti-IFNγ (both 2.5 μg/mL; R&D Systems),5 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 10 ng/mL; Sigma),ionomycin (5 nM; Merck Biosciences), and cyclosporin A (CsA;Calbiochem) were used as indicated. CD4+ T cells were culti-vated in RPMI 1640 (5, 6).

Preparation of T-Cell subsets. Human T-cell subsets were isolatedand stimulated as shown previously (7, 8). Murine CD4+CD25+

naturally occurring regulatory T cells (nTreg) and CD4+CD25−

conventional naïve CD4+ T cells (Tconv) were isolated (6) usingDynal Mouse T Cell Negative Isolation Kit (Invitrogen) followedby staining with anti-CD25-PE mAb and anti-PE MACS beadsenrichment (Miltenyi Biotech).

Cell Culture and Stimulations. Priming and restimulation of primaryT cells was performed using plate-bound anti-CD3 mAb (145-2C11,5 μg/mL) plus anti-CD28 mAb (37.51, 1 μg/mL) (both BD Phar-mingen). After 72 h the cells were washed and cultured on freshplates for additional 96 h. For intracellular cytokine analysis cellswere stimulated for 6 h with TPA/Iono in presence of GolgiPlugand GolgiStop (both BD Pharmingen).

FACS Staining. FACS staining (6) was carried out with the fol-lowing Ab: fluorescein isothiocyanate (FITC)-conjugated CD4(GK1.5), CD8α (53-6.7), CD19 (1D3), GITR (DTA-1); phycoer-ythrin (PE)-conjugated CD3ε (145-2C11), CD4 (RM4-5), CD19(1D3), CD25 (PC61), CD39 (24DMS1), CD73 (eBioTY/11.8)CD103 (2E7), CD107a (eBio1D4B), OX-40 (OX-86), Lag3(eBioC9B7W), CTLA-4 (UC10-4B9); allophycocyanin (APC)-con-jugated CD8α (53-6.7); eFluor710-conjugated GARP (YGIC86)and LAP (TW7-16B4); biotin-conjugated CD3ε (145-2C11), CD4(GK1.5), and CD90.2 (53-2.1), and secondary streptavidin-HRP,streptavidin-APC or streptavidin-PE mAb (all BD Pharmingen).Intracellular Foxp3 (FJK-16s, FITC-, PE-, and APC-conjugated),Helios (22F6, FITC-conjugated, BioLegend), and cAMP (SPM486,Abcam), together with donkey-anti-mouse-AlexaFluor555 (In-vitrogen), staining was performed using the Foxp3 staining kit(eBiosciences). Cytokine staining for IL-2-APC (JES6-5H4),IFN-γ–APC (XMG1.2), IL-17-PE (eBio17B7), and TNFα-PE(MP6-XT22) was performed using the IC Fixation Buffer Kit(eBioscience). Samples were analyzed on a FACS Calibur (BD

Biosciences) with CellQuest (BD Biosciences) and FlowJo soft-ware (TreeStar).

ELISA. Cells (1 × 106/mL) were cultivated as indicated for 48 h.The supernatant was analyzed by IL-2 ELISA (eBiosciences).

Calcium Measurement. Splenic CD4+ T cells (1 × 107) were in-cubated in medium containing 5% (vol/vol) FCS, 1 μM Indo-1-AM (Invitrogen), and 0.015% Pluronic F127 (Invitrogen) at 30°C for 25 min. The cell suspension was then diluted with 700 μLmedium containing 10% (vol/vol) FCS and incubated at 37 °Cfor another 10 min. The cells were washed twice with PBS fol-lowed by surface staining with anti-CD4-PacificBlue, anti-CD25-PE, and biotinylated anti-CD3ε (all eBioscience). For calciummeasurement, cells were diluted 1:10 in PBS containing 0.5 mMEGTA, and a baseline was recorded for 60 s. Ca2+ movementwas assessed after streptavidin-HPR cross-linking (eBioscience),followed by the addition of 1 mM Ca2+ after 3.5 min and Ionoafter 9 min of recording. After 12 min, another baseline of un-stimulated cells was recorded as control. Increases in free in-tracellular Ca2+ were measured in real-time on an LSR II (BD),and data were analyzed as median in comparative overlays withFlowJo software (TreeStar).

Proliferation Assays. Proliferation assay (6) was measured usinga Mach 2 Harvester (Tomtec).

Immunofluorescence. For confocal microscopy (5, 6) the followingprimary antibodies were used: anti-NFAT2 (7A6; BD Pharmin-gen), anti-NFAT1 (IG-209; immunoGlobe), anti-Smad3 (ab28379;Abcam), and anti-Foxp3 (FJK-16s; eBiosciences). Secondarystaining was performed using Abs: anti-rabbit Alexa-Fluor 647,anti-mouse Alexa-Fluor 488, and anti-rat Alexa-Fluor 555 (allMolecular Probes). Slides were mounted with Fluoromount-G(Southern Biotechnology) containing DAPI. Images were takenwith a confocal microscope (Leica TCS SP2 equipment, objectivelense; HeX PL APO, 40×/1.25–0.75) and LCS software (Leica).For statistics, more than 100 cells from at least three independentexperiments were counted, and mean fluorescence intensity percell was calculated.

Immunoblot. Protein lysate was made with radioimmunoprecip-itation assay buffer (RIPA) buffer andmeasured using bicinchoninicacid (BCA) reagent (BioRad). Equal amount of protein was frac-tionated by 8–12% SDS/PAGE and electroblotted on membranes(5). For detection, anti-NFAT2 (7A6; BD Pharmingen), anti-NFAT1 (IG-209; immunoGlobe), anti-NFAT4 (F-1; Santa CruzBiotechnology), and anti-β-actin (C4; Santa Cruz Biotech-nology) with anti-mouse or anti-rabbit peroxidase-coupled sec-ondary antibodies were used.

PCR and Quantitative RT-PCR. Genomic DNA was prepared withDNA-lysis buffer (including 0.2% SDS). PCR was performedusing the following primers (5′→3′): Nfat2 CCTATTTAAAC-ACCTGGCTCCCTGCG plus CCATCTCTCTGACCAACAG-AAGCC AG, Δexon3 CTAGGCCTCAGGCGTTCCACC plusCCTGCCTCTCTCAGCCTTTGA, Cebp CGAGCCACCGCG-TCCTCCAGC plus CCGGTCGGTGCGCGTCATTGC. RNAwas extracted using the RNeasy Micro Kit (Qiagen) followed bycDNA synthesis which was performed with the iScript II Kit(BioRad). Real-time quantitative RT-PCR was carried out withan ABI Prism 7700 detection system using the following primers:Nfat1 TCATAGGAGCCCGACTGATTG plus CCATTCC-

Vaeth et al. www.pnas.org/cgi/content/short/1203870109 1 of 16

www.pnas.org/cgi/content/short/1203870109

CATCTGCAGCAT, Nfat2 GATCCGAAGCTCGTATGGACplus AGTCTCTTTCCCCGACATCA, Nfat4 CACATCCCACA-GCCCAGTG plus CACATCCCACAGCCCAGTG normalizedto β-actin GACGGCCAGGTCATCACTATTG plus AGGAA-GGCTGGAAAAGAGCC.

ChIP Analysis. ChIP-IT Express kit (Active Motif) was usedaccording to the manufactures’ instructions, except for enzymaticshearing followed by additional sonication. The following IP-Abs were used: anti-NFAT2 (7A6; BD Pharmingen), anti-Foxp3(FJK-16s; eBioscience), and anti-Smad3 (Abcam). Primers (5′→3′)used were: Foxp3-Pr CTTCCCATTCACATGGCAGGC plusCTTTGCCCTTTACAAGTCATCTG; Foxp3-CNS1 GCACTTG-AAAATGAGATAACTGTTC plus CATCACAGTACATACGA-GGAAATG; and Foxp3-CNS3 CCAGATGGACGTCACCTACCplus GGCGTTCCTGTTT-GACTGTTTC.

Electromobility Shift Assay (EMSA). Nuclear proteins from humanand murine T-cell subsets were prepared and stored by ProteoJETKit (Thermo Scientific). hFoxp3-Prom-NFAT GTTCTTCTTCC-TTGTTTTTTTTT; mFoxp3-Prom-NFAT GACTTATTTTCCCT-CAGTTTTTTTTTT;mFoxp3-CNS1-NFATGCTTCATTTTTTCC-ATTTACTG; mIL2-Pubd CCCCAAAGAGGAAAATTTGTTT(boldface, NFAT consensus sites); anti-NFAT1 (D43B1, CellSignaling), and anti-NFAT2 (7A6, BD Pharmingen) were usedfor EMSA, which was performed as previously described (9).

Microarray. CD4+CD25+ nTregs and CD4+CD25− Tconv fromWT or Nfat1−/− × Nfat2fl/fl × Cd4-cre (DKO) mice were isolatedand stimulated for 24 h with plate-bound anti-CD3/28 in absence(Tconv and nTreg) or presence (iTreg conditions) of 5 ng/mLTGF-β, followed by RNA extraction using the standard TRIzolmethod (Invitrogen). Biotin-labeled amplified aRNA was pre-pared using the GeneChip 3′ IVT Express Kit and hybridizedto GeneChip mouse genome 430 2.0 arrays (Affymetrix) ac-cording to the manufacturer’s protocols. The trimmed meansignals of the probe arrays were scaled to a target value of 500 andexpression values determined using Affymetrix GeneChip Oper-ating Software. Data were analyzed and visualized as a heat mapusing GeneSpring GX 12.0 software (Agilent Technologies).The original microarray data can be found in the ArrayExpress

database under the accession no. E-MEXP-12345 (www.ebi.ac.uk/arrayexpress).

CD28SA Treatment. Treatment of mice with superagonistic CD28mAb D665 was performed as previously described (4). Mice re-ceived a single i.p. injection of 250 μg CD28SA D665 (Serotec) orPBS as control. After 60 h serum samples were obtained from tailvein. Spleen and LN cells were harvested on day 3 post injection.

Adoptive Transfer Colitis and Endoscopy. Colitis was induced inRag1−/−mice by injecting i.p. 2.5 × 105 Cd90.1+ (WT) and 2.5 × 105

Cd90.2+ (WT or Nfat2fl/fl × Cd4cre) CD4+CD62L+CD25− cells.Mice were anesthetized [100 μL of a mixture of 100 mg/mLKetavest (Pfizer) and Rompun (Bayer Healthcare) i.p.] and clini-cal symptoms [murine endoscopic index of colitis severity (ME-ICS): total range 0–15 points; colon translucency (0–3 points),presence of fibrin (0–3 points), mucosa granularity (0–3 points),vascular pattern (0–3 points), and stool (0–3 points)] were ana-lyzed with a high-resolution video endoscopic system (KarlStorz) (10).

Skin TransplantModel.To test alloantigen-induced or nTreg in vivo(11, 12) mice received 200 μg of anti-CD4 YTS177.9 mAb (Bio-Xcell) i.v. on day −28/−27. On day −27 the mice also received250 μL donor-specific blood transfusions (DST) from BALB/cmice. CD4+CD25+ T cells were FACS-sorted on day 0, andC57BL/6 Rag2−/− mice were reconstituted i.v. with 2 × 105

C57BL/6 CD4+CD45RBhi cells along with 5 × 105 CD4+CD25+

nTreg cells isolated from naïve or 2 × 105 CD4+CD25+ Tregcells from YTS177/DST-pretreated mice. Next day, BALB/c tailskin allografts were transplanted onto flanks of reconstitutedmice. Graft survival between groups was monitored and com-pared using the log–rank test. For transfer of in vitro-generatediTregs, naïve CD4+ CD25− T cells were cocultivated with BALB/cCD19+ B cells in the presence of IL-2, TGF-β, anti-IL-12, anti–IFN-γ, and the anti-CD4 YTS177 for 2 wk, with restimulationafter 1 wk. FACS-sorted CD25hi T cells were verified for Foxp3expression, and equal numbers were used in transplantation.

Statistical Analysis. Groups were compared with Prism software(GraphPad) using two-tailed Student’s t test.

1. Lee PP, et al. (2001) A critical role for Dnmt1 and DNA methylation in T celldevelopment, function, and survival. Immunity 15:763–774.

2. Ranger AM, Oukka M, Rengarajan J, Glimcher LH (1998) Inhibitory function of twoNFAT family members in lymphoid homeostasis and Th2 development. Immunity 9:627–635.

3. Wing K, et al. (2008) CTLA-4 control over Foxp3+ regulatory T cell function. Science322:271–275.

4. Gogishvili T, et al. (2009) Rapid regulatory T-cell response prevents cytokine storm inCD28 superagonist treated mice. PLoS ONE 4:e4643.

5. Nayak A, et al. (2009) Sumoylation of the transcription factor NFATc1 leads to itssubnuclear relocalization and interleukin-2 repression by histone deacetylase. J BiolChem 284:10935–10946.

6. Vaeth M, et al. (2011) Regulatory T cells facilitate the nuclear accumulation ofinducible cAMP early repressor (ICER) and suppress nuclear factor of activated T cellc1 (NFATc1). Proc Natl Acad Sci USA 108:2480–2485.

7. Becker C, et al. (2009) Protection from graft-versus-host disease by HIV-1 envelopeprotein gp120-mediated activation of human CD4+CD25+ regulatory T cells. Blood114:1263–1269.

8. Kubach J, et al. (2007) Human CD4+CD25+ regulatory T cells: proteome analysisidentifies galectin-10 as a novel marker essential for their anergy and suppressivefunction. Blood 110:1550–1558.

9. Berberich-Siebelt F, et al. (2000) C/EBPbeta enhances IL-4 but impairs IL-2 andIFN-gamma induction in T cells. Eur J Immunol 30:2576–2585.

10. Weigmann B, et al. (2008) The transcription factor NFATc2 controls IL-6-dependentT cell activation in experimental colitis. J Exp Med 205:2099–2110.

11. Karim M, Kingsley CI, Bushell AR, Sawitzki BS, Wood KJ (2004) Alloantigen-inducedCD25+CD4+ regulatory T cells can develop in vivo from CD25-CD4+ precursors ina thymus-independent process. J Immunol 172:923–928.

12. Sawitzki B, et al. (2005) IFN-gamma production by alloantigen-reactive regulatoryT cells is important for their regulatory function in vivo. J Exp Med 201:1925–1935.


http://www.ebi.ac.uk/arrayexpress

http://www.ebi.ac.uk/arrayexpress


Fig. S1. Conditional disruption of Nfat2 exon3 in mice. (A) Strategy for introduction of loxP sites into the mouse Nfat2 locus flanking exon 3. Genomic locus ofthe Nfat2 exon3 region (Upper) and the targeting vector containing a loxP sites flanked version of exon3 including a neomycin resistance cassette (Lower)bordered by frt sites. (B and C) Confirmation of conditional inactivation of Nfat2 in CD4+ T lymphocytes by CD4-mediated cre. (B) PCR analysis of genomicDNA of isolated CD4+ T cells with specific primers for the loss of Nfat2 exon3 (Δexon3) and total Nfat2 (wt Nfat2) of WT (Nfat2+/+ × Cd4-cre), heterozygous(Nfat2+/fl × Cd4-cre), and knockout (Nfat2fl/fl × Cd4-cre) mice. (C) Immunoblot of protein from CD4+ T cells of WT, heterozygous, and homozygous mice. Arrowsindicate isoforms of NFAT2. (D) Breeding of Nfat2fl/fl × Cd4-cre mice to Nfat1−/− mice resulted in NFAT1NFAT2 double-deficient T lymphocytes. Immunoblotanalysis of NFAT expression in total thymocytes from WT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− × Nfat2fl/fl × Cd4-cre mice. (E) Foxp3-specific inactivation of Nfat2 inTreg cells by Foxp3-IRES-cre (FIC). Immunoblot with proteins isolated from CD4+CD25− and CD4+CD25− T cells of WT and Nfat2fl/fl × FIC mice.



B

wild type

Nfat2fl/fl x Cd4-cre

A

5 ng/ml TGFβ 2.5 ng/ml TGFβ 1.25 ng/ml TGFβ 0.6 ng/ml TGFβ

45.0 43.7 37.6 19.2

36.1 22.5 17.0 6.9

Foxp3

IL-2 only

IL-2 & TGFβ

unstim. anti-CD3/28 anti-CD3/28+ 10 ng/ml CsA

3.3 2.4 7.4

5.3 71.5 9.3

wild type Nfat2fl/fl x Cd4-cre

Nfat1–/– x Nfat2fl/fl x Cd4-cre

C D

CD4

IL-2

TH0

TH1

TH17

iTreg

IL-17A

IFNγ

3.4

2.4

6.8

2.8

3.9

2.2

39.2

1.1

32.3

0.9

14.1

1.5

5.7

2.3

8.2

3.8

2.3

2.4

2.2

47.4

4.2

11.7

1.7

3.5


47.3 33.9

CD90.2+

27.6 20.3

CD4

IFNγ

32.6 22.1

CD4

TN

Fα

16.6 7.8

CD4

IL-1

7

Foxp3

CD

25

Fig. S2. Direct influence of NFAT on Foxp3 and cytokine induction. (A) TGF-β–mediated iTreg induction of WT CD4+CD25− T cells is abrogated in the presenceof 10 ng/mL CsA. (B) CD4+CD25− T cells from WT and Nfat2fl/fl × Cd4-cre mice were stimulated for 3 d with plate-bound anti-CD3/28 in the presence of IL-2 anddifferent concentrations of TGF-β, followed by rest of 4 d. Representative FACS analysis of CD4+CD25+Foxp3+ T cells in culture. (C and D) Impaired cytokineexpression in NFAT-deficient CD4+ T cells. (C) CD4+CD25− T cells from WT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− × Nfat2fl/fl × Cd4-cre mice were cultivated for 3 dwith plate-bound anti-CD3/28 under TH0- (IL-2, anti-IL-4, anti-IFNγ), TH1- (IL-12, IFNγ, anti-IL-4), iTreg- (IL-2, TGF-β, anti-IL-4, anti-IFNγ), and TH17-polarizingconditions (IL-6, IL-21, TGF-β, anti-IL-4, anti-IFNγ, anti-IL-2). Cells were restimulated for 6 h with TPA/Iono followed by intracellular IFN-γ and IL-17 analysis usingFACS. (D) Measurement of intracellular IL-2, IFN-γ, TNF-α, and IL-17 of CD90.2+ WT or CD90.2+ Nfat2fl/fl × Cd4-cre T cells transferred into Rag2−/− (lymphopenic-induced colitis, parallel to Fig. 2 B–D; Fig. S4). Total cells from mLN were stimulated for 6 h with TPA/Iono followed by intracellular FACS-staining; gating onCD4 and CD90.2 identified WT or Nfat2fl/fl × Cd4-cre adoptively transferred T cells.



H

mFoxp3-CNS1-NFATmIL2-Pubd mFoxp3-Prom. hFoxp3-Prom.

Tconv

TGFβnTreg

++–

*

anti-CD3/28+ –

+ ++ +– – –

+ – –

– – – –+++– + –

+ ++ +– – –

+ – –

– – – –+++– + –

+ ++ +– – –

+ – –

– – – –+++– + –

+ ++ +– – –

+ – –

– – – –+

Tconv

TGFβnTreg

anti-NFAT2

hFoxp3-Prom. mFoxp3-CNS1-NFAT

anti-NFAT1comp. hFoxp3-Prom.

+ + + –– – –– – – + + +

*

ss NFAT1–ss NFAT2–

+ + +

+ + ++ + ++ –– ––– –

–– ––– – –– – –

–– – –– – –– – – – –+– – + – – + – – + – – +–– + – – + – – + – – + ––

I

Tconv

TGFβnTreg

anti-NFAT2

EL-4

CsA

mFoxp3-CNS1-NFAT mFoxp3-Prom-NFAT

ss NFAT2–

+ +– –– –– –– –– +

– –– +

– –– +

– –– + – +

+ ++ + – –– – – –– – – –– –+ +

– – – –– – – –– – + + + + + +

+ +– –– –– –– –– +

– –– +

– –– +

– –– + – +

+ ++ + – –– – – –– – – –– –+ +

– – – –– – – –– – + + + + + +

J

wild ty

pe

Nfat2fl/f

l x Cd4

-cre

Nfat1–/– x

Nfat2fl/f

l x Cd4

-cre

Thymus

Spleen

LN

13.7 13.4 14.6

1.3 0.9 1.2

12.4 13.3 9.3

A B

wild ty

pe

Nfat2fl/f

l x Cd4

-cre

Nfat1–/–

x

Nfat2fl/f

l x Cd4

-cre

Foxp3

CD

25

Cwild

type

Nfat1–/–

Nfat1–/–

x

Nfat

4–/–

Thymus

Spleen

LN

Foxp3

CD

25

Thymus

Spleen

Lymph nodes%

CD

4+C

D25

+F

oxp3

+

0.6 0.7 1.4

14.0 10.5 11.3

9.1 8.6 10.3

E

Thymus

Spleen

mLN

Foxp3

CD

25

0.9 0.8

15.3 22.1

17.1 13.3

Spleen

% C

D4+

CD

25+

Fox

p3+

cel

ls

F

0

10

20

30

0

5

10

1520

25

0

0.5

1.0

1.5

2.0

2.5

0

5

10

15

20

25

0

5

10

15

20

25

wild type Nfat2fl/fl x FIC

wild type

Nfat2fl/fl x FIC

LN

Dwild type Nfat4–/–

0.8 0.6

15.414.7

13.7 15.9

Thymus

Spleen

mLN

Foxp3

CD

25

G

n.s.

rela

tive

expr

essi

on to

β-a

ctin

unstim. anti-CD3/28

Nfat2 Nfat1 Nfat4

nTreg Tconv Tconv+TGFβ



% C

D4+

CD

25+

Fox

p3+

%

CD

4+C

D25

+F

oxp3

+

0

1

2

3

0

1

2

3

0

1

2

3

Fig. S3. Normal numbers of nTreg in NFAT-deficient mice. (A and B) Nfat2fl/fl × Cd4-cre and Nfat1−/− x Nfat2fl/fl × Cd4-cre mice show normal frequencies ofCD4+CD25+Foxp3+ nTreg in thymus, spleen, and LN. (A) Representative FACS analysis of gated CD4+ cells and (B) synopsis of those from different lymphoid organs; eachsymbol represents one littermate. (C) AppearanceofnTreg is unchanged inNfat1−/−andNfat1−/−×Nfat4−/−mice comparedwithWT. FlowcytometryofgatedCD4+ cells inthymus, spleen,andLN. (D)Nfat4−/−miceshownormal frequenciesofCD4+CD25+Foxp3+nTreg in thymus, spleen,andLN, representativeFACSanalysisofgatedCD4+Tcells.(EandF) Treg-specificdeletionofNfat2viaFoxp3-IRES-cre (FIC) doesnotchangethenTregcompartment. (E) RepresentativeFACSdeterminationofCD4+CD25+Foxp3+cellsin thymus, spleen, and LN and (F) summary of four analyzed mice. (G) RNA of Nfat1, Nfat2, and Nfat4 is expressed in unstimulated and anti-CD3/28−stimulated Tconv,nTreg, and Tconv in presence of TGF-β (iTreg induction), detected after 24 h by real-time PCR and normalized to β-actin. Human (H and I) and murine (J) nuclear proteinextracts exhibit binding activity of NFAT1 and NFAT2 to Foxp3-CNS1, but hardly to the promoter (cells harvested for EMSA after 48 h; ss, supershift; *unidentified band).



Rag1–/–

2.5 x105 CD4+CD62L+CD25– cells:Cd90.2+ wild type or

Cd90.2+ NFAT-deficient

6 weeks

2.5 x105 CD4+CD62L+CD25– cells:Cd90.1+ congenic

wild type

A

Cd90.1+ wild type & Cd90.2+ wild type

Histology FACSCytokines

weight changeendoscopy

70

80

90

100

110

120

0

2

4

6

8

10

Cd90.1+ wild type & Cd90.2+ Nfat2fl/fl x Cd4-cre

B C

weeks after T cell transfer weeks after T cell transfer

0 1 2 3 4 5 6 1 2 3 4 5

D

100 x

400 x

wei

ght c

hang

e in

%

clin

ical

sco

re (

endo

scop

y)healthy control

Cd90.1+ wild type & Cd90.2+ wild type

Cd90.1+ wild type & Cd90.2+ Nfat2fl/fl x Cd4-cre

n.sn.s

Fig. S4. Normal course of lymphopenia-induced colitis by NFAT2-deficient T cells. (A) Schematic overview of adoptive T cells transfer to induce colitis. 2.5 × 105

CD90.2+CD4+CD62L+CD25− WT or NFAT-deficient T cells (Nfat2fl/fl × Cd4-cre or Nfat1−/− × Nfat2fl/fl × Cd4-cre) along with 2.5 × 105 CD90.1+CD4+CD62L+CD25−

congenic WT cells were injected i.p. into Rag1−/− recipient mice. (B–D) Normal course of colitis in Rag1−/− mice receiving NFAT2-deficient CD4+CD62L+CD25−

T cells. (B) Monitoring weight change and (C) the clinical symptoms by colon endoscopy over 6 wk revealed only marginal differences in the characteristicsof colitis in mice receiving either WT or Nfat2fl/fl × Cd4-cre T cells (five mice per group). (D) Representative H&E staining of paraffin-embedded colonsections showed no alteration of colitis in mice receiving either WT or Nfat2fl/fl × Cd4-cre T cells. As a reference a healthy colon (Left) in 100× and 400×magnification is given.



A

prol

ifera

tion

in c

pm (

x104

)

wild typeiTreg

Nfat2fl/fl x Cd4-creiTreg

CD4+ gatedFoxp3

CD

4

0

5

10

15

unstim

1 : 0.8

1 : 01 : 0

.71 : 0

.41 : 0

.31 : 0

.2

unstim

1 : 0.8

1 : 01 : 0

.71 : 0

.41 : 0

.31 : 0

.2


1 : 0.8

1 : 0.7

1 : 0.4

1 : 0.3

1 : 0.2

47.1 45.7

37.5 40.0

29.4 32.6

19.1 19.7

16.3 15.8B

Rag2–/– C57/BL6recipient

skin graftsurvival

CD45RBhigh T cells

BALB/c skin allograft

CD25+Foxp3+ iTreg

wild type or Nfat2fl/fl x Cd4-creC57/BL6 CD4+CD25– Tconv

+BALB/c CD19+ B cells +IL-2/TGFβ

+blocking anti-IL-12/IFNγ+ anti-CD4 mAb (YTS177, 1µg/ml)

day 14

day 0

CD

4+C

D25

+ iT

regs

in %


wild type

C

D

E

Nfat2fl/fl x Cd4-crewild type

Tconv only (n=5)

wild type iTreg (n=4)

Nfat2fl/fl x Cd4-cre iTreg (n=2)

graf

t sur

viva

l in

%

0.3

5.4

21.7

72.7

0.1

9.2

15.2

75.5

tota

l CD

4+C

D25

+ iT

regs

(x1

07)

0

1

2

3

4

5

0

20

40

60


wild type

p=0.0009 p=0.0388

0 5 10 15 20 25 300

20

40

60

80

100

days after transplantation

Foxp3

Hel

ios

2.8 1.4

92.5 92.53.4

0.3 1.4

5.7

CD4+CD25+

iTreg

CD4+CD25–

non-iTreg

Fig. S5. Normal in vitro and in vivo suppressive capacity of NFAT2-deficient TGF-β–induced Treg. (A) CD4+CD25− Tconv from WT and Nfat2fl/fl × Cd4-cre micewere stimulated for 3 d with anti-CD3/28 in presence of 5 ng/mL TGF-β followed by 12 d rest in medium containing IL-2 and TGF-β. CD25hi cells were enrichedand Foxp3 expression was analyzed by FACS. Different dilutions of Foxp3+ iTreg (Right) were cocultured together with WT responder CD4+CD25− T cells andAPCs for 3 d. Ratio of responder vs. iTreg cells in culture is denoted (unstim, unstimulated 1:0.8 ratio as control). After 48 h proliferation was measured bythymidine incorporation (Left). Data are mean ± SD of triplicates. (B–E) NFAT2-deficient iTreg are fully suppressive in vivo. (B) Scheme of experimental setup toexamine suppressive capacity of NFAT2-deficient iTreg in a model of allogenic skin transplantation. (C) CD4+CD25− Tconv from WT and Nfat2fl/fl × Cd4-cre mice

Legend continued on following page



were cocultivated for 2 wk with allogenic BALB/c splenocytes in the presence of IL-2, TGF-β, anti-IL-12, and anti-IFNγ. Frequency of CD4+CD25+ allogenic-induced iTreg was controlled by FACS (n > 5). (D) Foxp3 and Helios expression of WT and Nfat2fl/fl × Cd4-cre CD4+CD25+ iTreg from these cultures. (E) 2 × 105

WT or Nfat2fl/fl × Cd4-cre iTreg depicted in C and D were injected along with 2 × 105 CD4+CD45RBhi T cells i.v. in the Rag2−/− recipient mice and the BALB/c skinallograft survival was measured by log–rank test. All mice receiving only CD4+CD45RBhi cells acutely rejected their skin transplants [n = 5, mean survival time(MST) = 15 d]. Addition of Nfat2fl/fl × Cd4-cre iTreg prolonged skin graft survival as efficiently as WT CD4+CD25+ iTreg (WT: n = 4, MST = 22.0 d; Nfat2fl/fl × Cd4-cre:n = 2, MST = 27 d). Shown is one representative experiment out of two.



CD3ε

B22

0

B

CD4

CD

8α

C

0

100

200

300

IL-2

in p

g/m

l

*

42.6 54.1 56.4

26.9 16.7 12.5

20.2 26.1 29.3

47.5 43.2 41.6

p < 0.0005

p = 0.036

21.8

34

13.2

20.5

2.31

7.89

5.87

14

9.35

10.9

19.4

25.4

4.87

7.6

12.5

19.1

18.2

23.9

Spleen

LN

Spleen

LN

Thymus

wild ty

pe

Nfat2fl/f

l x Cd4-cr

e

Nfat1–/– x

Nfat2fl/f

l x Cd4-cr

e



wild type

Spleen Lymph nodes

wild

type

Nfa

t2fl/

fl x

Cd4

-cre

Nfa

t1–/

– x

Nfa

t2fl/

fl x

Cd4

-cre

Foxp3CD3ε

E



wild type

0

20

40

60

80

0

50

100

150

Spleen Lymph nodesto

tal c

ell n

umbe

r in

106

A D

tota

l cel

l num

ber

in 1

06

Fig. S6. NFAT-deficient mice display no evidence of autoimmune disorders. (A) Total cell numbers in spleen and LN of WT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− ×Nfat2fl/fl × Cd4-cre mice. Data are mean ± SD from at least six littermates. (B) Representative FACS analysis of B and T-cell compartments in spleen and LN ofWT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− × Nfat2fl/fl × Cd4-cremice. (C) Frequency of CD4+ and CD8+ T cells in thymus, spleen, and LN of WT, Nfat2fl/fl × Cd4-cre, andNfat1−/− × Nfat2fl/fl × Cd4-cre mice examined by flow cytometry. (D) Impaired IL-2 production by NFAT-deficient CD4+ T cells in vitro. Isolated CD4+CD25− Tconvfrom WT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− × Nfat2fl/fl × Cd4-cre mice were stimulated with plate-bound anti-CD3/28 for 48 h, and produced IL-2 was assayedfrom supernatants by ELISA. Data are mean ± SD of triplicates done in one experiment and representative of two; asterisk denotes nondetectable levels of IL-2in 100-fold diluted supernatants. (E) Paraffin-embedded section of spleen and LN from untreated WT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− × Nfat2fl/fl × Cd4-cremice were used for two color staining of Foxp3 (red) and CD3ε (green). (Scale bar, 80 μm.)



A C

B

thym

us p

opul

atio

n in

%

wild type Nfat1–/– xx x Nfat2fl/fl x Cd4-cre

D

E

2.782.95

0.78 1.03

7.3 71.3

14.0 4.7

6.1 55.9

31.4 6.4

4.6 2.5

4.3 1.6

0.7 0.5

90.4 95.7

CD4+ gated

SS

C

Foxp3

SS

C

CD8α

CD

4

CD8α

CD

4

DP CD4 CD8 DN0

10

20

304050607080

CD4 CD80

1

2

3

4

5

F

spl

een

popu

latio

n in

%

wild type

Nfat1–/– xx x Nfat2fl/fl x Cd4-cre

day 1

Thymus Spleen0

1

2

3

4

5

6

Thymus Spleen0.00.51.01.52.0

68

101214

day 1

day 12

day 1

day 12

day 1

day 12

day 1

day 12

0.390.26

10.3 10.0

11.6 49.6

34.1 4.6

8.7 82.7

5.6 3.0

3.1 0.2

93.7 3.0

2.6 0.1

94.5 1.9

CD

4+F

oxp3

+ i

n %

Thymus Spleen0

20

40

60

80

CD4 CD80

1

2

3

4day 12

day 1 day 12

DP CD4 CD8 DN0

10

20

305060708090

Thymus Spleen 00.20.40.60.81.0

6

8

10

12

tota

l cel

l num

ber

(x10

6 )

day 1 day 12

day 1 day 12

**

*

*

*

*

**

**

Fig. S7. Regular frequency of NFAT1 plus -2 double-deficient nTreg in newborn and young mice. (A) Representative FACS analysis of CD4/CD8 cell distribution(Upper) and nTreg frequency among CD4+ cells (Lower) in the thymus of newborn and 12-d-old WT and Nfat1−/− × Nfat2fl/fl × Cd4-cre mice. (B) RepresentativeFACS analysis of CD4/CD8 cell distribution (Upper) and nTreg frequency among CD4+ cells (Lower) in the spleen of newborn and 12-d-old WT and Nfat1−/− ×Nfat2fl/fl × Cd4-cremice. (C–F) Quantification of at least three independent experiments shown as frequency in % (C–E) and total cell number (F) of thymus andspleen. *P ≤ 0.05; **P ≤ 0.005.



0

10000

20000

30000

40000

0

10000

20000

30000

40000

50000

0

10000

20000

30000

40000

50000

A

B

unstim

0 : 11 : 0 1 : 11 : 0

.5

1 : 0.25

1 : 0.125

unstim

0 : 11 : 0 1 : 11 : 0

.5

1 : 0.25

1 : 0.125

unstim

0 : 11 : 0 1 : 11 : 0

.5

1 : 0.25

1 : 0.125

0

5000

10000

15000

unstim

1 : 0 1 : 11 : 0

.5

1 : 0.25

0

5000

10000

15000

20000

25000

unstim

1 : 0 1 : 11 : 0

.5

1 : 0.25

Nfat1–/– nTreg Nfat1–/– x Nfat4–/– nTreg

wild typecontrol

Nfat2fl/fl x FIC+CD28SA

Spleen Lymph nodes

wild type+ CD28SA

D

C

Ser

um IL

-2 in

pg/

ml

prol

ifera

tion

in c

pm

prol

ifera

tion

in c

pm

14.0 14.8

25.2 21.3

24.5 25.5

0

50

100

150

200

250

300

Foxp3

CD

25

E

C57/BL6donor

Rag2–/– C57/BL6recipient

skin graftsurvival

CD45RBhigh T cells

BALB/c skin allograft

CD25+ nTreg

wild typeor


*0 20 40 60 80 100

0

20

40

60

80

100

graf

t sur

viva

l in

%

survival days

Tconv only

wild type nTregNfat2fl/fl x Cd4-cre nTreg

Fig. S8. NFAT-deficient nTreg exhibit normal suppressive capacities in vitro and in vivo. (A) CD4+CD25+ nTreg from WT, Nfat2fl/fl × Cd4-cre, and Nfat1−/− ×Nfat2fl/fl × Cd4-cre (Upper) or Nfat1−/− and Nfat1−/− × Nfat4−/− mice (Lower) were stimulated together with WT responder CD4+ T cells and APCs for 3 d. Ratioof responder vs. nTreg in culture is denoted (unstim, unstimulated 1:1 ratio as control). After 48 h proliferation was measured by thymidine uptake. Data aremean ± SD of triplicates done in one experiment and representative of four. (B) Frequency of Nfat2fl/fl × FIC nTreg is not impaired in spleen and LN of micetreated with 250 μg superagonistic CD28 mAb D665 (CD28SA) for 3 d. (C) Serum IL-2 was assayed by ELISA 2.5 h after CD28SA injection. Data are mean ± SD oftriplicates. (D and E) NFAT2-deficient nTreg fully prevent skin allograft rejection in an adoptive transfer mouse model. (D) Schematic overview of adoptive




transfer of B6 WT or Nfat2fl/fl × Cd4-cre CD4+CD25+ nTreg along with CD4+CD45hi T cells into a B6 Rag2−/− recipient mouse, receiving a BALB/c skin allograft. (E)5 × 105 nTreg along with 2 × 105 CD4+CD45RBhi cells were injected i.v. and BALB/c skin allograft survival in the Rag2−/− recipient mouse was measured by log–rank test. All mice receiving only CD4+CD45RBhi cells acutely rejected their skin transplants (n = 5, MST = 12.8 d). Addition of WT (n = 4, MST = 51.0) or Nfat2fl/fl

× Cd4-cre Treg (n = 5, MST = 60.4) prolonged skin graft survival.



Fig. S9. Analysis of NFAT-deficient Treg on mRNA and protein levels. (A and B) Analysis of molecules involved in Treg-mediated suppression and homeostasis.(A) Freshly isolated splenic CD4+ T cells from WT or Nfat1−/− × Nfat2fl/fl × Cd4-cre (DKO) mice were stained for indicated molecules. Subsequent intracellularFoxp3 staining identified nTreg and Tconv using FACS; i.c., intracellular; s, surface staining. (B) CD4+CD25+ nTreg and CD4+CD25− Tconv from WT or Nfat1−/− ×Nfat2fl/fl × Cd4-cre (DKO) mice were isolated and stimulated for 3 d with plate-bound anti-CD3/28 in absence (Tconv and nTreg) or presence (iTreg conditions)of TGF-β. Denoted molecules on Foxp3− Tconv, Foxp3+ nTreg, or TGF-β–induced iTreg were analyzed by FACS. (C) Flow cytometry of CD25 expression in variousNFAT-deficient Treg populations. CD4+CD25+ nTreg and CD4+CD25− Tconv from WT, Nfat2fl/fl × Cd4-cre, Nfat1−/− × Nfat2fl/fl × Cd4-cre, Nfat1−/−, and Nfat1−/− ×




Nfat4−/− mice were isolated and stimulated for 3 d with anti-CD3/28 in the presence of TGF-β. After 4 d the CD25 expression on Foxp3+ nTreg and iTreg wasmeasured by FACS. (D and E) Analysis of genes potentially regulated by NFAT:Foxp3-complexes using microarray gene profiling. (D) Venn diagram illustrationsof genes regulated more than twofold between Nfat1−/− × Nfat2fl/fl × Cd4-cre (DKO) and WT in CD4+CD25+ nTreg and CD4+CD25− Tconv stimulated for 24 hwith anti-CD3/28 in presence (Tconv+TGF-β, iTreg polarizing conditions) or without TGF-β (nTreg and Tconv). Data represent two completely independentbiological experiments. (E) Representation of suggested NFAT:Foxp3-regulated genes in DKO and WT nTreg, Tconv, and Tconv+TGF-β stimulated for 24 h withanti-CD3/28 as heatmap (Left) or bar graphs (Right). Clustering analysis and heatmap of expression values show the log2 transformed expression intensity ofthe genes. Genes regulated ≥twofold between DKO and WT are tagged with an asterisk. Data from two independent biological experiments are given as anaverage of reliable entities representing denoted genes.



% n

ucle

ar N

FAT

2

0

20

40

60

80

100

0

20

40

60

80

100

CD4+ T cells w/o TGF CD4+CD25– T cells + TGF

Foxp3+Foxp3–Foxp3+Foxp3–

p < 0.0001 p < 0.0001

CD4+CD25– Tconvw/o stimulation

CD4+CD25+ nTregw/o stimulation

CD4+CD25– Tconvanti-CD3/28

CD4+CD25+ nTreganti-CD3/28

NFAT2 Foxp3 DAPI

D

C

A

B

NFAT2 Foxp3 Smad3 DAPI

CD4+CD25–

Tconv

CD4+CD25+

nTreg

w/o

TGFβ

CD4+CD25–

Tconv

CD4+CD25–

iTreg

0.0

0.5

1.0

1.5

nTregTconv

w/o stimulation anti-CD3/28

ratio

nuc

leus

/cyt

osol

NFA

T2

fluor

esce

nce

sign

al p =0.002

% n

ucle

ar N

FAT

2

E F

0 0.5 1 1.5 2 4 6 8 16 24 480

20

40

60

80

100

time (h) on plate-bound anti-CD3/28

cells

with

nucl

ear

NFA

Tc1

( %

)

G

0 h

0.5 h

1 h

1.5 h

2 h

4 h

6 h

8 h

16 h

24 h

48 h

Indo1-A

M (

viole

t/blu

e)

CD4+CD25– TconvCD4+CD25+ nTreg

time (min)

CD3e crosslink

1 mMCaCl2

Iono baseline

CD4+ neg. isolated cells

CD

25

CD4

CD4+CD25– TconvCD4+CD25+ nTreg

NFAT2 Foxp3 DAPINFAT2DAPI

Fig. S10. Impaired nuclear translocation of NFAT2 in Foxp3+ cells. (A and B) Four-color staining of NFAT2 (red), Foxp3 (yellow), Smad3 (blue), and chromatin(cyan) of freshly isolated CD4+ conventional T cells (Tconv) and CD4+CD25+ nTregs stimulated for 6 h with anti-CD3 and anti-CD28 (Upper). Foxp3− Tconv andTGF-β-induced Foxp3+ iTregs generated from CD4+CD25− Tconv after 24 h stimulation with anti-CD3 plus anti-CD28 (Lower). (B) Quantification of nuclear andcytosolic fluorescence signal of NFAT2 in freshly isolated CD4+ Tconv and CD4+CD25+ nTregs by confocal microscopy, nuclei were demarcated by DAPI staining.The ratio of nuclear/cytosolic signal was calculated of unstimulated (w/o) and 6 h anti-CD3/CD28 stimulated cells, data are mean ± SEM from at least 100 cellsout of three individual experiments. (C) Histograms of one representative Foxp3− Tconv and nTreg cell, respectively, show different NFAT2 nuclear trans-location after 6 h stimulation by anti-CD3/28. Red line represents NFAT2 staining, yellow line Foxp3, and the nucleus is demarcated by cyan DAPI staining.(D) Quantification of cells with nuclear NFAT2 in CD4+ Tconv and CD4+CD25+ nTreg (Left) and 24 h TGF-β–induced Foxp3+ iTreg (Right), data are mean ± SEM




from more than 100 cells out of four individual experiments. (E and F) CD4+ T cells were stimulated with plate-bound anti-CD3/28 for the indicated time points,and NFAT2 nuclear translocation was recorded using confocal microscopy. Representative pictures (E) and the quantification of more than 30 individual cells(F) are shown. (G) Calcium-influx measurement in CD4+CD25− Tconv (red line) and CD4+CD25+ nTreg cells (blue line) by real-time flow cytometry. CD4+ T cellswere loaded with calcium-sensitive dye Indo1-AM and stained subsequently with anti-CD25-PE and biotinylatd anti-CD3ε. After 1 min the CD3ε Abs were cross-linked with streptavidin followed by the addition of 1 mM CaCl2 after 3.5 min. As a positive control Iono was added after 9 min of measurement, as a negativecontrol nonactivated cells were analyzed again as a baseline after 12 min.



supporting information - pnas · elisa. cells (1 × 106/ml) were cultivated as indicated for 48 h....

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