Supplementary Fig.

a CD4+CD25+

LN Spleen
21.3 27.4
Events

Blimp1-GFP

b c
25 16
Foxp3 expression (x 103)

20
12
Tgfb1 expression
15
8
10

4
5

0 0
CD25+ CD25- CD25+ CD25-
GFP- GFP+ CD62L- CD62L+ GFP- GFP+ CD62L- CD62L+

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 2

a gfp/gfp (CD4+CD25+)
b gfp/gfp (CD4+CD25+)
75.3 18.9 48.8 2.4 Spleen Lymph node

GFP- 95.1 96.4

Events
CD62L
CD44

5.8 0.02 31.5 18.4

33.1 7.9 70.6 17.6

GFP+ 96.1 92.8

Events
CD69

GITR

48.3 10.7 10.3 1.5

25.3 8.1 28.7 24.5 Foxp3

CD38
IL-7R

58.1 8.4 44.7 2.0

Blimp1-GFP

c Ly5.1 : Blimp1gfp/gfp (CD4+) CD4+Foxp3+

+/+ (Ly5.1+) gfp/gfp (Ly5.2+) +/+ (Ly5.1+) gfp/gfp (Ly5.2+)
11.1 5.7 9.2 4.3 24.2 8.9 15.8 19.7
CD103
CD25

70.6 12.6 73.3 13.1 57.4 9.5 43.6 21.0

CTLA4 ICOS

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 3

a b 4.0
3.0

3.5
*
2.5

gfp/gfp Treg contribution (Log2)

Il10 expression (x103)

3.0

2.0 2.5

2.0
1.5 * *
1.5
1.0
1.0 *
0.5
0.5 n.s.

0 0.0
p

p
gf

gf

gf

gf
p/

p/

p/

p/

-0.5
+

+
+/

+/

+/

+/
gf

gf

gf

gf

LN spleen PP IEL Lung

CD25+ CD25+ CD25- CD25- -1.0
GFP+ GFP- CD62L- CD62L+

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 4

a
CD4+
PBS IL-2c
20.0 39.8
Events

Foxp3

b c IL-2 IL-2 + IL-4

IL-2 IL-2 + IL-4
78.9 89.7
Events
Events

IL-2 + IL-6 IL-2 + IL-12 IL-2 + IL-6 IL-2 + IL-12

79.1 79.1
Events
Events

Blimp1-GFP Foxp3

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 5

a b
CD4+ CD4+
mes LN Spleen mLN lung
13.8 7.1
Tbx21+/+ 5.31 1.15 4.94 0.73
Tbx21+/+
Blimp1+/gfp

91.5 1.90 90.8 3.51

Tbx21-/- 5.26 0.92 6.56 1.24 15.4 6.7

Tbx21-/-
Blimp1+/gfp
CD25

Foxp3
92.0 1.81 89.6 2.55

Blimp1-GFP CD4

c CD4+ Foxp3+ CD4+ Foxp3+

Tbx21+/+ Tbx21-/-
mLN lung mLN lung
18.8 7.51 26.5 9.15 19.6 8.8 21.7 5.36
CD103

22.7 50.9 29.0 35.3 28.6 43.0 43.1 29.8

CD62L CD62L

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 6

a spleen (CD4+)
+/+ Irf4-/-
14.4 0.7 6.8 0.01

Foxp3

81.6 0.3 93.2 0.01

KLRG1

b +/+ Irf4-/- Blimp1fl/flLck-Cre

(Ly5.1+) (Ly5.2+) (Ly5.2+)
39.7 0.3 0.7 0 19.5 0
T-bet

47.5 12.6 53.1 46.2 38.2 42.3

30 18.9 50.9 17.9 22.8 37.8

CD62L

49.9 1.2 30.6 0.5 37.8 1.7

CFSE

c CD4+
Tbx21+/+ Tbx21+/- Tbx21-/-
15.5 0.66 16.0 0.27 15.3 0.31
Foxp3

75.1 8.71 76.9 5.85 82.8 1.56

T-bet

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 7

a b Ly5.1 : Blimp1gfp/gfp
Ly5.1 : Blimp1+/+
CD4+Foxp3+ CD4+Foxp3+
20.6 20.5 7.8 4.2
+/+ mLN 70 +/+
Ly5.1 gfp/gfp
60

Ki67+ (% of Foxp3+)
50

9.8 49.1 51.5 36.6 40

25.8 35.4 9.9 8.6

+/+ 30
Lung
Ly5.2
20
CD103

10
Ki67

7.0 31.8 73.8 7.8

spleen LN lung
Bcl2 Ly5.1

c d Ly5.2+
CD4+ CD4+CD25+
43.7 21.1 40
Bim+/+ 14.5 2.1 Bim+/+ P=0.0006
Blimp1gfp/+ Blimp1gfp/+ 35
Blimp1-GFP+ (% of Tregs)
30

25
83.0 0.5 34.0 1.1
20

Bim-/- 22 6.1 Bim-/- 38.8 37.6

15
Blimp1gfp/+ Blimp1gfp/+
10

5
CD25

ICOS

70.3 1.7 21.2 2.4

+
-/-
+/
m
m

Blimp1-GFP Blimp1-GFP
Bi
Bi

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 8

a b
Ccr6 Klrg1
Il10

Blimp1
IRF4fl/+ / IRF4fl/-

c
IRF4fl/+ / IRF4fl/-

Blimp1+/gfp / Blimp1gfp/gfp

Blimp1+/gfp / Blimp1gfp/gfp

d
Blimp1 Ebi3 Ccr6 1.5
Rorc
2.0 2.0 4
Expression (x 102)

1.5 1.5 3
1.0

1.0 1.0 2

0.5
0.5 1.5 1

0 0 0 0
4+
4+

4+
4+
eg

eg

g
eg
g

g
g

D
D

e
D
D
re

re

re
re
Tr

Tr

Tr
Tr

C
C

C
C
-T

-T

-T
-T
+

ve

+
+

ve
ve
ve
FP

FP

FP

FP

FP

FP
FP

FP
ai

ai
ai
ai
G

G
G

N
N
N

200 Tcf7 80 Id3 10 Bcl2

8
e CD4+CD25+
Expression (x 102)

150 60
0.4 2.5
6
100 40
4
KLRG1

50 20
2

86.6 10.5
0 0 0
4+

4+

4+
eg

eg

eg
g

Blimp1-GFP
D

D
re

re

re
Tr

Tr

Tr
C

C
-T

-T

-T
+

+
e

ve

ve
FP

FP
FP

FP

FP

FP
v
ai

ai

ai
G

G
G

G
N

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 9

a CD4+
12.1 5.1
Foxp3

81.9 0.9

CD103

b +/+ Irf4-/- Irf4-/- Irf4-/-

empty vector empty vector Blimp1 vector IRF4 vector w/o
0.8 0.4 0.25 0.5 0.01
Foxp3

CD4

29.8 45.5 68 1.9 53.4 1.6 26.7 21.9

CD103

18.3 6.4 30.1 0 43.7 1.2 42.0 9.4

KLRG1

69.6 10.7 22.8 75.0

Events

ICOS

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 10

0.08
Irf4-/-

Il10 occupancy by IRF4 (% of input)

+/+
Blimp1fl/fl Lck-Cre
0.06

0.04

0.02

0.00
Pr S9

Pr S9

Pr S9
C S3

C S3

C S3
om

om

om
N

N
N

N
C

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 11

a
Il10 locus, intron 1

putative putative
IRF4 sites Blimp1 sites

mouse
mouse

human

human

b
Il10 locus, intron 1

IRF4 IRF4
Blimp1 Blimp1 IRF4
Blimp1 Blimp1 Blimp1

mouse
human
90 100 110 120 130 140 150 160 170 180 190

IRF4
Blimp1

mouse
human
200 210 220 230 240 250 260 270 280 290 300

Nature Immunology doi:10.1038/ni.2006

 Supplementary Fig. 12

Nature Immunology doi:10.1038/ni.2006

 Supplementary Table 1
Microarray analysis of GFP+ Treg cells from Blimp1gfp/gfp
and Blimp1+/gfp mice. Data is the fold change (Blimp1gfp/gfp / Blimp1+/gfp).

FDR

Nature Immunology doi:10.1038/ni.2006

 Supplementary Table 2
Analysis of Blimp1-differentially expressed genes for their dependence on IRF4.
Data shows fold changes of these genes in the IRF4fl/- / IRF4fl/+ microarray
analysis (based on Zheng et al., 2009)17.
FDR

Nature Immunology doi:10.1038/ni.2006

 Supplementary Material and Methods

RT-PCR primer sequences used for mRNA expression analysis:

Hprt: 5’- GGGGGCTATAAGTTCTTTGC-3’

5’- TCCAACACTTCGAGAGGTCC-3’

Foxp3: 5’- GGCCCTTCTCCAGGACAGA-3’

5’- GCTGATCATGGCTGGGTTGT-3’

Tgfb1: 5’- CAACGCCATCTATGAGAAAACC-3’

5’- AAGCCCTGTATTCCGTCTCC-3’

Il10: 5’- GAAGACCCTCAGGATGCGG-3’

5’- CCTGCTCCACTGCCTTGCT-3’

Tbx21: 5’- AGGGGACACTCGTATCAACAGA-3’

5’- AGGGGGCTTCCAACAATG-3’

Tcf7: 5’-AGCTTTCTCCACTCTACGAACA-3’
5’-AATCCAGAGAGATCGGGGGTC-3’

Id3: 5’-CTCTTAGCCTCTTGGACGACAT-3’
5’-GATCGAAGCTCATCCATGCCCT-3’

Ccr6: 5’-CCTGGGCAACATTATGGTGGT-3’
5’-CAGAACGGTAGGGTGAGGACA-3’

Rorc: 5’-TTTGGAACTGGCTTTCCATC-3’
5’-AAGATCTGCAGCTTTTCCACA-3’

Bcl2: 5’-TTATAAGCTGTCACAGAGGGGCTAC-3’
5’-GAACTCAAAGAAGGCCACAATCCTC-3’

RT-PCR primer sequences used for ChIP analysis:

Il10 CNS3 5’-AGTTGAGCCATTAGGGTTGGATAGAGG-3’

5’-ACCCCAGGCAGAAGTCAGTCTAATATC-3’

Nature Immunology doi:10.1038/ni.2006

 Il10 CNS9 5’-CTTGAGGAAAAGCCAGCATC-3’
5’-TTTGCGTGTTCACCTGTGTT-3’

Il10 Prom 5’-GGCACCAGAACTCTCCTCTG-3’

5’-TGGGTTGAACGTCCGATATT-3’

Il10 PromA 5’-GACCTGGGAGTGCGTGAATGG-3’

5’-AGCGCTAAAGAACTGGTCGGAATG-3’

Il10 PromB 5’-TGCAGTAAGTACAGGTCACAGTCA-3’

5’-AGTTTTCTACCAGCAGCAAGCACA-3’

Il10 intron1 5’- CCCTCTTCTCATTCGTTTTCCA-3’

5’-CCAGGAAAGAATTTGAGAAAAATCA-3’

Ccr6 5’-GAACTCAGGATGGCGTGACTGGT-3’
5’-TCAATAACCCAGAGCACCGAGAA-3’

Cd19 3’ 5’- CTCGCCTAGCCTTTCTTC-3’

5’- TTCGGATGTGCCTGGGTTTAG-3’

Blimp1 3' 5’-ACCGTTGAAAGACGGTGACT-3’

5’-AATGGCCCACATCATCTGTG-3’

Blimp1 CNS9 5’-AAGCTGCTAAGTGGGAGAGT-3’

5’-GCATAATTTAGCGTTTGGTG-3’

Blimp1 Prom 5’-GCATGAGAGGCAGGGCAACA-3’

5’-CCTGCTAACTGAATACATTCAG-3’

Nature Immunology doi:10.1038/ni.2006