File name: Supplementary Information Description: …10.1038/s41467-017... · a b H2d H2b c WT...

Description of Supplementary Files

File name: Supplementary Information Description: Supplementary figures, supplementary table 1 and supplementary references. File name: Peer Review File

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Supplementary Figure 1a

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Supplementary Figure 1b

Supplementary Fig. 1 Defective polarization of lytic granules and mitochondria in NFATc1-

deficient CTLs. Bright field and fluorescence microscopy of living cells showing the recruitment of

lytic granules (green) and mitochondria (red) in WT (a) and Nfatc1-/- (b) CTLs upon contact with

aCD3/CD28-loaded beads. Arrows indicate the contacts between beads and CTLs. (c) Intensity

profile plots for lytic granules (LysoTracker® Green) and mitochondria (MitoTracker® Deep Red)

from WT and Nfatc1-/- CTL contacting beads loaded with aCD3/CD28. Left: Distribution of

organelles in WT CTLs. Right: Distribution of organelles in Nfatc1-/- CTL (3 to 5 profiles per time

point, ~10 cells each condition). Black bars indicate bead-cell border (bead size 5 µm).

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Intensity Profile Plot NFATc1 -‐/-‐ MitoTracker Red (n=29)Bead size ~5 µm

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Intensity Profile Plot WT MitoTracker Red (n=31)Bead size ~5 µm

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Intensity Profile Plot WT LysoTracker Green (n=17)Bead size ~5 µm

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Intensity Profile Plots NFATc1 -‐/-‐ CTL Lysotracker Green (n=52)Bead size ~5 µm

c

3

wild type

Nfatc2 -/-

Nfatc1-/-

Nfatc1-/-

Nfatc2 -/-

GzmB DAPI merge

10µm

wild type

Nfatc1-/-

15

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GzmB (pg/ml x 103)

2015

10

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vesicles per cell

wild type

Nfatc1-/-

Nfatc2 -/-Nfatc1-/-

Nfatc2 -/-

a b

+

Supplementary Fig. 2 Effect of NFATc1 ablation on the accumulation of granzyme B in CTLs. (a)Accumulation of granzyme B-containing cytoplasmic granules in Nfatc1-/- CTLs. Confocalmicroscopy. (b) Left, column presentation showing results from two independent assays. Right,granzyme B secretion, measured by ELISAs from the supernatant of CTLs. Typical assays of 3experiments are shown as means ± SEM.

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a

b H2bH2d

c

WT Nfatc1-/-n.s.2.0

1.5

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CD44+CD62L-

CD44+

Total flux (photons/sec.) x108

IL-17 Granzyme-B Perforin

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IFN-gamma IL-2 TNFIFN-gamma

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IL-17

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Granzyme-B

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NC

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MFI

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FI (I

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MFI

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MFI

(Fas

L)

0

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3000

4000 *

MFI

(CD

69)

MFI

CD62L CD44 CD25 FasL CD69d

e

Supplementary Fig. 3 Cytotoxic activity and surface marker expression of Nfatc1-/- CTLs. (a)Cytotoxic activity of sorted in CD62L+CD44+ and CD62L-CD44+ CTLs. CTLs were sorted accordingto the surface markers CD62L and CD44. The sorted cells were incubated for 4 h with MOPC 315plasmacytoma cells expressing a luciferase indicator gene. A decreased chemiluminiscence (Totalflux) indicates cytotoxic activity. Data of 3 assays are shown.(b) Scheme of the generation ofallogeneic reactive CTLs in vitro for cytotoxicity and degranulation assays (presented in Fig. 2 b).(c) Expression of lymphokines, granzyme B and perforin 1 in and MFI values of CTLs (see Fig.2c). NC,negative control. (d) Expression of several surface markers and MFI values of CTLsgenerated for 6 d in vitro. ns, non-significant. (e) MFI values of cells shown in Fig. 2f. UnpairedStudent’s t-test was used. Data are shown as means ± SEM.

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ß-Act

a

50 kb29.210 kb 29.220 kb 29.230 kb 29.240 kb 29.250 kb

Cdkn1aNR_036469[0-50]

[0-50]

[0-25]

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

1 2 3 4 5 6 108 97 11 12 13

55

70

100

35

kD

25

NFATc1-alpha-spec. Ab

kD

10070

NFATc1-7A6-mAb

c

Fold induction

aCD8+ T cells

Cdkn1a

Fold induction

CTL-

Cdkn1a

Fold induction

CTL+

Cdkn1a


b

70

90

1

kD

2 3 4 5 6 7 8 9 10ß-Act

ß-ActNfatc1

Nfatc1f/fxCD4-cre

Nfatc1f/fxCD4-creWT WT

d

Supplementary Fig. 4 Degradation of NFATc1/aA and NFATc1-mediated control of Cdkn1a geneexpression encoding the cell cycle inhibitor p21WAW/CIP1. (a) Degradation of NFATc1/aA by proteinextracts from CTLs by incubation in vitro. Protein from KT12 NK cells overexpressing NFATc1/aAwas incubated for 30 min at 370 C alone (lane 1), with 30 mg nuclear protein from aCD8+Ts (lane3), from aCD8+Ts treated with 100 ng/ml rapamycin (lane 5), from CTLs treated for 2 d byaCD3/CD28 and 6 d by IL-2 (lane 7), from CTLs treated for 2 d by aCD3/CD28 and 6 d by IL-2followed by 5 h CsA (100 ng/ml) (lane 9), from CTLs treated for 2 d by aCD3/CD28 and 6 d by IL-2 and for 5 h by T+I (lane 11), or from NFATc2-deficient CTLs treated for 2 d by aCD3/CD28 andfor 6 d by IL-2 and for 5 h by T+I (lane 13). In the even lanes, the protein extracts of T cells alonewere fractionated. The immune blot was incubated subsequently with Ab raised against theNFATc1 a-peptide, or with the 7A6 NFATc1 Ab. One typical blot of 3 assays is shown. (b) Immuneblots of nuclear proteins from CD8+T cells of WT and Nfatc1fl/fl x CD4-cre mice. Cells were leftunstimulated (lane 1) or stimulated with aCD3/CD28 for 6 h (lane 2), 3 d (lanes 3 and 5), or withaCD3/CD28 for 3 d followed by incubation with IL-2 for 2 d (lanes 4 and 6) or 7 d (lanes 7-10). Inlanes 8 and 10, CTLs were finally stimulated by T+I for 5 h. (c) Real-time PCR assays of Cdkn1aRNA. RNAs were isolated from aCD8+Ts, and from non-induced CTLs (CTL-) and CTL+ cellsstimulated by T+I for 5 h. Two-tailed unpaired Student’s t-test was used. Data are shown asmeans ± SEM. (d) Below, binding of NFATc1/A-Bio and NFATc21 to the Cdkn1 locus. ChIP seqdata are shown from CTL+ cells.

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a

b aCD8+ T cells

Fold induction

Gzmb Prf1


ns4

3

2

1

0

200

150

100

50

0

Fold induction

[0-50]

[0-50]

[0-25]

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

Gzmb

56.860 kb 56.870 kb 56.880 kb 56.890 kb 56.900 kb 56.910 kb60kb

[0-50]

[0-50]

[0-25]

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

II12rb2

67.280kb123kb

II123r

67.300kb 67.320kb 67.340kb 67.360kb 67.380kb

[0-50]

[0-50]

[0-25]

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

MAST3

73.320kb51kb

Arrdc2II12rb1

73.330kb 73.340kb 73.350kb 73.360kb

Supplementary Fig. 5 NFATc1 binding to Il12 beta receptor loci, and to the granzyme B locus. (a)Binding of NFATc1/A-Bio and NFATc21 to the Il12rb and Il12rb2 genes encoding IL-12 receptorproteins. ChIP seq data of CTLs stimulated for 5 h by T+I. (b) Above, real-time PCR assays ofGzmb and Prf1 RNAs encoding the effector molecules granzyme B and perforin 1, respectively.Data of 5 PCR assays are shown, relative to naïve WT CD8+T cells and normalized to Actb. Two-tailed unpaired Student’s t-test was used. Data are shown as means ± SEM. RNAs were isolatedfrom aCD8+Ts. Below, binding of NFATc1/A-Bio and NFATc21 to the Gzmb gene. ChIP seq data ofCTLs stimulated for 5 h by T+I.

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[0-50]

[0-50]

[0-25]

Refseq genesBirA

Nc1A-Bio.BirA

Nc2

49kb98.990 kb 98.000 kb 98.010 kb 98.020 kb 98.030 kb

Ccr7

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

[0-50]

[0-50]

[0-25]

70kb9.450 kb9.440 kb 9.470 kb9.4640 kb 9.490 kb9.480 kb 9.4500 kb

II7r

97kb

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

[0-50]

[0-50]

[0-25]

30.810 kb 30.820 kb 30.830 kb 30.840 kb 30.850 kb 30.860 kb 30.870 kb 30.880 kb 30.890 kb

Irf4sp22

Refseq genesBirA

Nc1A-Bio.BirA

Nc2

[0-50]

[0-50]

[0-25]

99kb52.070 kb 52.080 kb 52.090 kb 52.100 kb 52.110 kb 52.120 kb 52.130 kb 52.140 kb 52.150 kb

Tcf7p1a

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

[0-50]

[0-50]

[0-25]

60kb72.900 kb 72.910 kb 72.920 kb 72.930 kb 72.940 kb 72.950 kb

ItgaeItgae Gsg24J11Rik

Supplementary Fig. 6 Binding of NFATc1/A-Bio and NFATc21 to the Ccr7, Il7r, Itgae, Tcf7 andIrf4 loci encoding the chemokine receptor Ccr7, the IL-7 receptor a chain, the integrinCD103/integrin aE, and the transcription factors Tcf1 and Irf4, respectively. ChIP seq data ofCTLs stimulated for 5 h by T+I.

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Refseq genesBirA

Nc1A-Bio.BirA

Nc2

81.260 kb157kb

81.280 81.300 81.320 81.340 81.360 81.380

[0-50]

[0-50]

[0-25]

Actn1

Fold induction

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Actn1

CTL- CTL+

Nfatc2 -/-

Refseq genesBirA

Nc1A-Bio.BirA

Nc2

16.850 kb99kb

16.860 16.870 16.880 16.890 16.900 16.910 16.920 16.930

[0-50]

[0-50]

[0-25]

PlekEldr Fbxo48

Fold induction

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Plek

Refseq genesBirA

Nc1A-Bio.BirA

Nc2

13.470 kb

Vim

73kb13.480 13.490 13.500 13.510 13.520 13.530

[0-50]

[0-50]

[0-25]

Trdmt1

Fold induction 54

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Vim nsWT Nfatc1 -/-

Gene GeneNfatc2 -/-(-)

Nfatc1-/-(-)

Nfatc2 -/-(+)

Nfatc1-/-(+)

Nfatc2 -/-

Nfatc1-/-

globalglobal

ActbDnm1FlnaFlnbPsrc1

Tubb6

Actn 1Plek

Tubb5

Tubb2bTubb2a

Tubb4bTubb4a

Vim

Tuba1aTuba1bTuba1cTuba4a

ActbDnm1FlnaFlnbPsrc1

Tubb6

Actn 1Plek

Tubb5

Tubb2bTubb2a

Tubb4bTubb4a

Vim

Tuba1aTuba1bTuba1cTuba4a

b

a

aCD8+Ts CTLs

Supplementary Fig. 7 Expression of genes affecting the re-modeling of cytoskeleton in Nfatc1-/-and Nfatc2-/- CD8+T cells. (a) Heat map of RNA expression in aCD8+Ts (left) and CTLs (right) ofselected genes encoding cytoskeleton proteins. NGS transcriptome data of Nfatc1-/- and Nfatc2-/-aCD8+Ts and CTLs are shown, relative to WT cells. (b) Left, binding of NFATc1/A-Bio and NFATc21to the Vim, Plek and Actn1 loci in CTL+ cells. ChIP seq assays. Right, real-time PCR assays ofVim, Plek and Actn1 RNAs encoding vimentin, pleckstrin and a-actinin 1, respectively. RNAs wereisolated from CTLs. Data of 5 PCR assays are shown, relative to naïve WT CD8+T cells andnormalized to Actb. Two-tailed unpaired Student’s t-test was used. Data are shown as means ±SEM. 10

GlucoseSlc2a3/Glut3Slc2a1/Glut1

Hk2

Gpi

Pfkm

Gapdh

Aldoc

Pgk1

Eno1

Pkm

Ldha

Pgm2

Glucose 6-P

Fructose 6-P

Fructose 1,6-bis-P

Glyceraldehyde 3-P

1,3-Biphosphoglycerate

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

Pyruvate

Lactate

WT Nfatc1 -/- Nfatc2 -/-

Supplementary Fig. 8 Effect of NFATc1 and NFATc2 ablation on the RNA expression of genes of aerobic glycolysis in aCD8+T cells. NGS results of transcriptome assays.

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Supplementary Fig. 9 Effect of NFATc1 ablation on metabolism of CTLs. (a) Heat map of RNAexpression of mitochondrial genes in aCD8+T cells, compared to WT cells. For comparison, theexpression of nuclear cytochrome c-1 gene (Cyc1) is also shown. (b) Immune blot showing the

12

effect of aCD3/CD28, IL-2 and Rapamycin on the Thr37/46 phosphorylation of the mTOR target4E-BP1 in splenic WT and Nfatc1-/- CD8+T cells upon treatment for 24 h. (c) Heat map of RNAexpression of genes of glycolysis cascade in Nfatc1-/- and Nfatc2-/- CTL- and CTL+ cells. (d)Incorporation of 2-NBDG into CTL- cells upon incubation for 1 h at 37oC. One typical assay of 2experiments is shown. (e) Real-time PCR assays of Slc2a1, Slc2a3 and Hk2 RNAs encoding theglucose transporters Glut1 and Glut3, and hexokinase 2, respectively, isolated from CTL- andCTL+ cells. Data of 5 PCR assays are shown, relative to naïve WT CD8+T cells and normalized toActb. (f) Extracellular flux analysis. 4x105 CTL- or CTL+ cells were seeded in a plate pre-coatedwith poly-D-lysine (Sigma;; 50 mg/ml) and subjected to extracellular flux analysis. Typical assays of3 experiments are shown. For (e), two-tailed unpaired Student’s t-test was used. Data are shown asmeans ± SEM.

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Fold induction 8

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Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

[0-50]

[0-50]

[0-25]

23kb61.812 kb

Myc

61.816 kb 61.820 kb 61.824 kb 61.828 kb 61.832 kb

125.104

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

24kb125.108 125.112 125.116 125.120 125.124

GapdhIffo1 Ncapd2[0-50]

[0-50]

[0-25]

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

113kb82.660 82.680 82.700 82.720

Hk2[0-50]

[0-50]

[0-25]

82.740

Refseq genes

BirA

Nc1A-Bio.BirA

Nc2

124,410 124,430

Slc7a5

[0-50]

[0-50]

[0-25]

BC048644

124,400 124,42043kb

Supplementary Fig. 10 NFATc1 binding to the Hk2, Gapdh, Slc7a5 and Myc loci encodinghekokinase 2, glyceraldehyde 3-phosphate dehydrogenase, cationic aa transporter Slc7a5 and c-Myc, respectively. (a) ChIP seq assays of NFATc1/A-Bio and NFATc21 binding to the Hk2, Gapdhand Slc7a5 genes in CTL+ cells. (b) Above, ChIP seq assays of NFATc1/A-Bio and NFATc21binding to the Myc locus in CTL+ cells. Below, real time PCR assays of Myc RNA. Data of 5 PCRassays are shown, relative to naïve WT CD8+T cells and normalized to Actb.Two-tailed unpairedStudent’s t-test was used. Data are shown as means ± SEM. 14

1) Martinez, G. J. et al. The transcription factor NFAT promotes exhaustion of activated CD8(+) T cells. Immunity 42, 265-278, doi:10.1016/j.immuni.2015.01.006 S1074-7613(15)00032-1 [pii] (2015).

Reference

15

Gene Primer Sequence

Ifng For: 5’-‐GAGCTCATTGAATGCTTGGC Rev: 5’-‐GCGTCATTGAATCACACCTG

Cxcr3 For: 5’-‐TCTCGTTTTCCCCATAATCG Rev: 5’-‐AGCCAAGCCATGTACCTTGA

Ccr7 For: 5’-‐GTCTCTCTCCAGCTAGCCCA Rev: 5’-‐CAAACAGGAGCTGATGTCCA

Cdkn1a For: 5’-‐ACGGGACCGAAGAGACAAC Rev: 5’-‐CAGATCCACAGCGATATCCA

Slc2a3 For: 5’-‐: ATCGTGGCATAGATCGGTTC Rev: 5’-‐CCGCTTCTCATCTCCATTGT

Cdkn2a For: 5’-‐GCAGAAGAGCTGCTACGTGA Rev: 5’-‐CGTGAACATGTTGTTGAGGC

Cad For: 5’-‐TACGCAGTTCTCATCGACCA Rev: 5’-‐TGGGAGTTGCATGAAGAGTG

Myc For: 5’-‐ACGGAGTCGTAGTCGAGGTC Rev: 5’-‐AGAGCTCCTCGAGCTGTTTG

Sell For: 5’-‐TTCATGGCTTTCCTTTCACA Rev: 5’-‐CTGGCATTTCTCATTTGGCT

Gls2 For: 5’-‐AGTTCACCACGGCTCTGAAG Rev: 5’-‐ CACACCTGGATCCCAGACAC

Slc1a5 For: 5’-‐GGACGTCTTTCATCTCCACAA Rev: 5’-‐ACTCCTTCAATGATGCCACC

Cdkn1b For: 5’-‐GGGGAACCGTCTGAAACATT Rev: 5’AGTGTCCAGGGATGAGGAAG

Il7r For: 5’CATTTCACTCGTAAAAGAGCCC Rev: 5’-‐TGGAAGTGGATGGAAGTCAA

Ccl3 For: 5’-‐GTGGAATCTTCCGGCTGTAG Rev: 5’-‐ACCATGACACTCTGCAACCA

Ccl4 For: 5’-‐GAAACAGCAGGAAGTGGGAG Rev: 5’-‐CATGAAGCTCTGCGTGTCTG

Il2 For: 5’-‐CGCAGAGGTCCAAGTTCATC Rev: 5’-‐AACTCCCCAGGATGCTCAC

Itgae For: 5’-‐GCCCAGTCCACATCCATATT Rev: 5’-‐GCTGCATCTGCTCCAGCTAT

Slc2a1 For: 5’-‐GAGTGTGGTGGATGGGATG Rev: 5’-‐AACACTGGTGTCATCAACGC

Ccr7 For: 5’-‐ACACAGGAAGGCTGTGCTTT Rev: 5’-‐CATGGACTGCTATCTGCGTC

Actn1 For: 5’-‐TCGGAAGTCCTCTTCGATGT Rev: 5’GGGAGAAGCAGCAGAGGAAG

Gzmb For: 5’-‐ CATGTAGGGTCGAGAGTGGG Rev: 5’ CCTCCTGCTACTGCTGACCT

Prf1 For: 5’-‐ TGGAGGTTTTTGTACCAGGC Rev: 5’ TAGCCAATTTTGCAGCTGAG

Eomes For: 5’-‐ GACCTCCAGGGACAATCTGA Rev: 5’ GGCCTACCAAAACACGGATA

Hk2 For: 5’-‐ GGAACCGCCTAGAAATCTCC Rev: 5’-‐ GGAGCTCAACCAAAACCAAG

Tbx21 For: 5’-‐ ATCCTGTAATGGCTTGTGGG Rev: 5’-‐TCAACCAGCACCAGACAGAG

Irf4 For: 5’-‐ CAAAGCACAGAGTCACCTGG Rev: 5’ TGCAAGCTCTTTGACACACA

Plek For: 5’-‐ CCACATGGGTTTCCAGGTAT Rev: 5’-‐ AAGAGTGGACCCGTGTGTCT

Vim For: 5’-‐ TCCACTTTCCGTTCAAGGTC Rev: 5’-‐ AGAGAGAGGAAGCCGAAAGC

Supplementary Table 1

List of Real Time-PCR Primers

16

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