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Supplementary text and figuresSupplementary Figures 1-12
Control of regulatory T cell and Th17 cell differentiation by inhibitory helix-loop-helix protein Id3
Takashi Maruyama1,* Jun Li1,*, Jose P. Vaque2, Joanne E. Konkel1, Weifeng
Wang3, Baojun Zhang4, Pin Zhang1, Brian Zamarron1, Dongyang Yu3, Yuntao
Wu3, Yuan Zhuang4, J. Silvio Gutkind2 & WanJun Chen1
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Supplementary Fig. 1
CD69 CD45RB
CD44 CD62L
Relative
cellnumber
Id3/Id3+/+
a b
0
5
10
15
20
25
3 4 12 21 42 112Age (d)
CD
4+Foxp3+(
%)
Id3+/+
Id3/
* **
*********
27
32 33
17 12
20Relat
ivecellnumber
Spleen LN Thymus
Id3+/+
Id3/
c
0
7
14
21
28
35
Ki67+(
%)
P = 0.03
Id3+/+ Id3/0
15
30
45P = 0.014
Id3+/+ Id3/
Ki67+(
%)
0
10
20
30 P = 0.04
Id3+/+ Id3/
Ki67+(
%)
d
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Supplementary Fig. 1. Defective generation and suppressive function ofTreg cells in Id3
/ mice. a, Kinetics of CD4+Foxp3+ T cells in the spleens of
mice.Data represent mean s.d. of Treg cells in individual mice. Each timepoint contains three to five mice (Id3+/+) or four to eight mice (Id3/) except at
day 42 with two mice in each group. * P< 0.05; ** P< 0.01; *** P< 0.001.
b, Flow cytometry analysis of surface markers (CD44, CD69, CD45RB andCD62L) on splenic T cells in symptomatic Id3/mice (>4-6 months)
compared to age-matched WT mice. Data are shown as histograms ofindicated markers in gated CD4+ T cells and represent at least threeexperiments.
c,d, Frequency of Ki67+ dividing CD4+Foxp3+ Treg cells in the spleen, lymphnodes (LN) and thymus in 3-5-month-old mice. c, Flow cytometry of Ki67+
frequency within gated CD4+Foxp3+ Treg cells in each tissue in one mouserepresentative of four (Id3+/+) and three (Id3/); d, Percent Ki67+ Foxp3+ cells
(mean s.e.m.) in the spleen, LN or thymus in the mice in c. P
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0
20
40
60
80
100
120
plus Id3+/+ Treg
Plus Id3/ Treg
1:0 1:0.25 1:0.5 1:1
3Huptake(CPMx
103)
Id3/ CD4+CD25 to Treg cells ( ratio)
Supplementary Fig. 2
a
b
Med
60 43Id3+/+ 25 13
CD3 + Treg (x104)
5 2.5 1.25 0.625
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0
10
20
30
40
50
60
0 0.02 0.2 2 20
Id3+/+
Id3/
TGF- (ng/ml)
CD4+CD25+Foxp3+(%)
Supplementary Fig. 3
Supplementary Fig. 3. Foxp3+ Treg generation in Id3/T cells in response
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0
10
20
30
Id3+/+
Id3/
Norm
alizedSmad7
mRNA
a
b
Supplementary Fig. 4
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Id3
GAPDH
CD4+CD25 CD4+CD25+
Supplementary Fig. 5
b
0
0.4
0.8
1.2
1.6
2
CD4+CD25
CD4+
CD25+
P= 0.002
N
ormalizedId3mRNAa
c
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 1 2 6 24
Time (h) after TCR stimulation
Med
TGF-
NormalizedId3mRNA
d
0
0.5
1
1.5
2
2.5
2h 24h
MedTGF-
Time (h) after TCR stimulation
( Smad3/ T cell)
NormalizedId3mRNA
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E-47
-actin
TGF-
IL-6
+ + + +
+ + + +
Id3+/+
Id3/
Supplementary Fig. 6
a b
0
5
10
15
Med TGF- Med TGF-Tgfbr1f/f
CD4-cre+Tgfbr1f/+
CD4-cre+
RelativeE2A
bindingat
Foxp3promoter
c
00.2
0.4
0.6
0.8
1
1.2
CD25- CD25+
RelativeE2A
bindingat
F
oxp3promoter
bHLH
loxP loxP
JW1 JW2
JW1 JW2
bHLHflox
bHLHdel
d
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Supplementary Fig. 6. E2A binding at the Foxp3 promoter in TGF-receptorI deficient T cells and in freshly isolated Id3/Treg cells.
a, E47 protein (immunoblot) in CD4+CD25T cells 2h after activation with anti-CD3 and CD28 mAb and indicated cytokines.
b, Analysis of relative E2A binding (E47/control IgG) to the Foxp3 promoter inCD4+CD25 T cells from TGF-b receptor I deficient (Tgfbr1f/fCd4-Cre+) orlittermate control (Tgfbr1f/+ Cd4-Cre+) mice in response to TCR stimulation with
or without TGF- (24h). Data are displayed as mean s.d. of the values oftriplicate wells in one experiments representative of two. The mean value ofE47/control IgG in control T cells treated with anti-TCR mAb alone was set as 1.
c, Analysis of E2A binding (E2A/IgG control, ChIP-qPCR assay at +327/+513 ) to
the Foxp3 promoter in CD4+
CD25
(CD25
) or frshly isolated CD4+
CD25+
Treg(CD25+) cells isolated from spleen and lymph nodes in Id3/mice (3 weeks-old,
cells pooled from 11 mice). Data are displayed as mean s.d. of the values oftriplicate wells. The mean value of E47/control IgG in control CD4+CD25 T cells
was set as 1. d,e,Efficient depletion of HEB and E2A after tamoxifen treatment.HEBf/fE2Af/fER-Cre mice were i.p. injected with tamoxifen (1mg/day) orsunflower seed oil for 5 consecutive days. Analysis of deletion level for
splenocytes is determined by PCR. Primer design designated in above diagrams
for flox and deleted alleles. d, HEB deletion. Primers JW1 and JW2 identify floxand deleted alleles (Wojciechowski, J.et al, J Immunol178, 5717-5726 (2007).e, E2A deletion. Primers neo for and YZ198 detect the flox allele (Lazorchak,A S t l J I l 177 2495 2504 (2006)
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Supplementary Fig.7
TGF-
0.5 0.5 41
2
0.5 60
0.4
31
35
2
49
1.7
29
1.4
68
0.3
Med TGF-,IL-4 TGF-,IFN- TGF-,IL-4,IFN-
p3
Id3+/+
Id3/
b
44 41
4 6
Foxp3
CD25
TGF- TGF-+IL-6a
Id3+/+
Id3/
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Supplementary Fig. 8
100 101 102 103 104
IL13-PE
092809.PBS.WT.Spl.#1.005
092809.PBS.Id3.Spl.#1.007
0.4
3
4
2
7
100
101
102
103
104
IFNg-PE
092809.PBS.WT.spleen.#1.007
092809.PBS.Id3.spleen.#1.009
32
29
0.5
0.03
100 101 102 103 104
Gata3-PE
092909.PBS.WT.Spl.#1.007
092909.PBS.Id3KO.Spl.#1.009
1
0.1
0.5
3
oxp3
Id3+/+
Id3/
d
a
Relativeil17a
expression
0
1
2
3
4
5
Scram SiRNA
Relative
rortexpression
***
Med
TGF- +IL-6
0
1
2
3
4
5
Scram SiRNA
***
0
10
20
30
40
50
Med TGF-b
IgG
E2A
Rela
tiveE2Abinding
atR
ORtpromoter
Med TGF-
b c
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0
10
20
30
40
IL-4(BAL
,pg/ml)
Id3+/+ Id3/
**
a
4
8
12
16
IL-4(plasma,pg/ml)
b
Supplementary Fig. 9
*
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Supplementary Fig. 10
0
10
20
30
40
50
WT KOId3+/+ Id3-/-
PlasmaIgE(ng/ml)
0
1
2
3
4
5
WT KOId3+/+ Id3-/-
LavageIgE(ng/ml)a
b Id3+/+ Id3-/-
35 1.1
1.6
18 0.7
1IL
-17
IL-13
9.6 0.5
4 4
3.5 0.5
1 4IFN-
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Supplementary Fig. 11. Th17, Th1, Th2 and Foxp3+ Treg cells in spleen and
LNs in asthmatic mice. a-f, Cells are from the draining LNs (Mediastinal).
a,c,e, Flow cytometry analysis of intracellular IL-17, IL-13, IFN- and IL-4cytokines or Foxp3+ Treg cells in CD4
+ T cells. Numbers in quadrants indicate
percent positive cells. Each plot is of one mouse representative of ten (Id3/)and nine (Id3+/+) mice. b,d,f, Percent of positive cells (mean s.d.) in the
mice in a,c,e respectively. g-i, Flow cytometry analysis of IL-17
+
(g) , IL-13
+
(j)and IFN-+ (i) positive cells and Foxp3+ cells (h) in CD4+ T cells in spleens inthe same mice. *, P< 0.05; **, P< 0.01; ns, not statistically significant.
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WT T cell Id3/ T cell
Opened
Foxp3 promoter
TGF-
receptor
Id3 E2A
E2A
IL-4
TCR
Gata3
IL-4
receptor
Rort
promoter
E2A E2AE2A
Smad2/3
?
Smad2/3
Closed
Foxp3 promoter
TGF-
TGF-
receptor
E2A
IL-4
E2A
TCR
Gata3
IL-4
IL-4
receptor
Gata3
E2A
Id3
IL-17
Smad2/3
E2A
Smad2/3
a bTGF-
TGF-TGF-
Smad2/3Smad2/3
Supplementary Figure 12
E2ARort
promoter
E2A?
15
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Supplementary Fig.12. A proposed model for Foxp3 gene transcription. a , WT CD4+ T cells. TGF- together with TCR
stimulation induces Foxp3 expression through at least two indispensable and complementary molecular events: by promoting E2A
protein binding to the Foxp3 promoter to positively regulate the gene transcription (enhancer) and by inhibiting/removing the negative
factors bound to the Foxp3 promoter such as GATA-3 (silencer).
TGF- enrichment of E2A binding at the foxp3 promoter could be mediated by, 1), a direct yet undetermined TGF- effect; 2), anindirect effect by suppression of Id3 expression at 12-24 h after TCR stimulation, which help break down the Id3-E2A complex; and/or
3), an inhibition of GATA-3 expression.
TGF- mediated GATA-3 suppression could be, 1), a direct effect by TGF- signaling; 2), an inhibition of IL-4 production and/or3), an
indirect effect by upregulation of Id3 expression at 1-2 h after TCR stimulation, which helps form the Id3-E2A complex that may
inhibit IL-4 production.
The activated Foxp3 promoter and consequent gene transcription could inhibit Rortgene activation. Although TGF- may induce
some degree ofRortpromoter activity through enriching E2A, the overall balance is toward against the optimal Rortgene activation
and IL-17 production in the absence of proinflammatory cytokines (e.g. IL-6) in wild type mice.
b. Id3/
CD4+
T cells. In the absence of Id3, TCR stimulation causes extremely higher level of GATA-3 expression, and consequentlylarge amounts of GATA-3 bind to the Foxp3 promoter in CD4+ T cells. This high level of GATA-3 is attributed largely to the
uncontrolled endogenous IL-4 production in these knockout T cells. IL-4 in turn induces GATA-3. Thus, Id3 is required for the control
of IL-4 production and consequent GATA-3 expression. How Id3 deficiency results in uncontrolled IL-4 remains unknown, but it could
involve a possible dysregulation of TCR signaling and/or the more Id3-E2A complex derived free E2A that could promote IL-4 gene
activation. In addition, the upregulated GATA-3 may also enhance more IL-4 production forming a positive feedback loop. As a result,
TGF- signaling fails to inhibit GATA-3 expression to the levels of WT T cells. The extra GATA-3 preoccupies the foxp3 promoter,
which precludes/interferes with TGF- mediated E2A binding at the foxp3 promoter. Although Id3/T cells may have slightly higher
levels of basal E2A binding at the foxp3 promoter due to the reported feature of Id3 as an inhibitor for E2A binding to its target genes,
E2A binding to the Foxp3 promoter cannot be enhanced enough to reach the threshold to initiate Foxp3 gene transcription in
response to TGF-, and Foxp3 gene cannot be transcribed (inactive).
Along with defective Foxp3 induction, Id3/T cells, show optimal Rortgene transcription and IL-17 production in response to TGF-in the absence of exogenous IL-6. Although the exact molecular mechanisms remain to be elucidated, at least two possibilities can
be participated. Firstly, the lack of Foxp3 expression could allow Rortgene activation in Id3/T cells; Secondly, TGF- mediated
E2A binding at the RORgtpromoter, together with other yet determined factors (molecules) that normally require IL-6 signal, may
directly promote the gene transcription ofRortand Il17in Id3/T cells.
Solid arrows (dark blue) indicate positive regulation; red lines indicate negative regulation;Dotted lines indicate no experimental
evidence available; question markers indicate to be determined.
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