nature immunology

8/8/2019 nature immunology

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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


2/17

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


4/17

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)


a

b

Med

60 43Id3+/+ 25 13

CD3 + Treg (x104)

5 2.5 1.25 0.625


5/17

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. Foxp3+ Treg generation in Id3/T cells in response


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0

10

20

30

Id3+/+

Id3/

Norm

alizedSmad7

mRNA

a

b



7/17

Id3

GAPDH

CD4+CD25 CD4+CD25+


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


8/17

E-47

-actin

TGF-

IL-6

+ + + +

+ + + +

Id3+/+

Id3/


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


9/17

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)


10/17

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/


11/17


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


12/17

0

10

20

30

40

IL-4(BAL

,pg/ml)

Id3+/+ Id3/

**

a

4

8

12

16

IL-4(plasma,pg/ml)

b


*


13/17


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-


14/17


15/17

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


17/17

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.

16

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