PBS OVA IP CP0

100

200

300

400

500

IL1

0 (

pg

/ml)

**

PBS OVA IP CP0

1000

2000

3000

4000

IFN

γ (p

g/m

l)

**

PBS RW IP CP0

1000

2000

3000

IL10

(pg

/ml)

***

dc

a b

NS RW IP CP0

500

1000

1500

2000

IFN

γ (

pg

/ml)

PBS

NS

*** ***

*** **

Supplemental Figure 2. Levels of IL10 and IFNγ are reduced in splenocyte cultures from STAT6-IP vaccinated, allergen sensitized mice. Splenocytes from IP, CP or sham (PBS) vaccinated and allergen sensitized and challenged mice were cultured in triplicate in the presence of OVA (a,b) or ragweed (c,d) and IL10 and IFNγ levels were determined from supernatants as described in Methods. (e) IDO expression was also increased in cells from IP-vaccinated mice as assessed by RT-PCR. Experiments were repeated at least 3 times. *p < 0.05, **p < 0.01, and ***p < 0.001.

PBS OVA IP CP0

10

20

30

IDO

/GAP

DH

****

e

IP-PBS DCsCP-PBS DCsOVA splen - DCs

*** ***

*** **

a b c

+

+++

-- +

+

++

-- +

+++

--

+

+++

-- +

+++

-- +

+++

--

NS

NS

de f

Supplemental Figure 3. Coculture of T or DCs cells from IP-vaccinated allergen naïve animals also modulate cytokine profiles. Mice were vaccinated as described in the methods but were not sensitized to allergen. (a-c) CD4+ T cells were isolated from IP or CP-vaccinated, non-sensitized (IP-PBS-T cells) mice and co-cultured with OVA sensitized CD4+T cell-depleted splenocytes in the presence of OVA and cytokine expression assessed by ELISA. Similarly, (d-f) DCs were isolated from IP (IP-PBS-DCs) or CP (CP-PBS-DCs) vaccinated non-sensitized mice and co-cultured with OVA sensitized DC-depleted splenocytes in the presence of OVA. Supernatants were collected, IL4, IL13, and TGFβ were determined. IL4 levels were unchanged in IP-T and DC co-cultures. IL13 levels were significantly lower in IP-T and DC cell co-cultures with OVA sensitized splenocytes associated with a marked elevation of TGFβ. *p < 0.05, **p < 0.01, and ***p < 0.001.

IP-PBS T cellsCP-PBS T cellsOVA splen -T cells

IL4

(pg/

ml)

IL13

(pg/

ml)

TG

Fβ (p

g/m

l)

IL4

(pg/

ml)

IL13

(pg/

ml)

TG

Fβ (p

g/m

l)

c d ***

IL1

0 (

pg/

ml)

a ***

IP-OVA T cellsCP-OVA T cellsOVA splen - T cells

IFNγ

(pg/

ml)

b ***

IP-OVA DCsCP-OVA DCsOVA splen - DCs

+

+++

-

- +

+++

-

-

+

+++

-- +

+++

--

Supplemental Figure 4. Co-culture of T cells or DCs from STAT6-IP vaccinated, allergen sensitized animals with T or DC-depleted splenocytes from allergic mice modulates cytokine profiles. (a-b) CD4+ T cells were isolated from IP-vaccinated and OVA sensitized/challenged (IP-OVA-T cells) or CP-vaccinated and OVA sensitized/challenged (CP-OVA-T cells) mice and co-cultured with T-depleted splenocytes from OVA sensitized/challenged animals in the presence of OVA. Similarly, (c-d) CD11c+ DCs were isolated from IP (IP-OVA-DCs) or CP (CP-OVA-DCs) animals as described above and co-cultured with OVA sensitized (OVA) DC-depleted splenocytes in the presence of OVA. Supernatants were collected, IL10, and INFγ was determined using ELISA. All cultures were performed in triplicate and experiments replicated at least 3 times. ***p < 0.001.

IL1

0 (

pg/

ml)

IFNγ

(pg/

ml)

***

a b c

PBS OVA IP-T cells CP-T cells0

100

200

300

TGFβ

(p

g/m

l)

***

PBS OVA IP-T cells CP-T cells0

1000

2000

3000

4000

5000

IL13

(pg

/ml)

***

PBS OVA IP-T cells CP-T cells0

100

200

300

400

500

IL4

(pg/

ml)

***

PBS OVA IP-T cells CP-T cells0

200

400

600

800

IL10

(pg

/ml)

***

PBS PBSOVA

OVA

IP-T cells CP-T cells

CP-T cells

CP-T cells0

500

1000

1500

2000

2500

IFN

γ (p

g/m

l)

PBS IP-T cellsOVA

***e

0

20

40

60

80

100

IDO

/GA

PD

H

***d f

CP-T cells

Supplemental Figure 6. Splenocytes from IP-T recipient mice show modulation in cytokine expression following sensitization and challenge. Naïve recipients were adoptively transferred CD4+ T cells from IP-vaccinated mice as described in the methods and recipient mice sensitized and challenged with OVA. Splenocytes were harvested, cultured with OVA and cytokine expression in supernatants assessed by ELISA. IP-T cell recipient animals showed reduced levels of IL4 (a), IL10 (b), IL13 (c) and IFNγ (d) while TGFβ (e) was significantly elevated. (f) IDO expression was also increased in IP-T-recipient cultures as assessed by RT-PCR. ***p < 0.001.

***

*** ***

*** ***

***

PBS OVA IP-DCs CP-DCs0

100

200

300

400

IL1

0 (

pg

/ml)

PBS OVA IP-DCs CP-DCs0

1000

2000

3000

IFNγ (

pg

/ml)

PBS OVA IP-DCs CP-DCs

**

d e

0

50

100

150

IDO

/GA

PD

H

***

a b

PBS OVA IP-DCs CP-DCs0

200

400

600IL

4 (p

g/m

l)

PBS OVA IP-DCs CP-DCs0

50

100

150

200

250TG

Fβ

(p

g/m

l)***

PBS OVA IP-DCs CP-DCs0

1000

2000

3000

4000

5000

IL13

(p

g/m

l)

***

c***

f*

*** *** ***

*** *****

Supplemental Figure 7. Splenocytes from IP-DC recipient mice show modulation in cytokine expression following sensitization and challenge. Naïve recipients were adoptively transferred DC cells from IP-vaccinated mice and then were sensitized and challenged with OVA. Recipient splenocytes were harvested and cultured with OVA and cytokine expression in supernatants assessed by ELISA. IP-DC cell recipient animals showed reduced levels of IL4 (a), IL10 (b), IL13 (c), IFNγ (d) while TGFβ (e) was significantly elevated. (f) IDO expression was also increased in IP-DC-recipient cultures as assessed by RT-PCR. *p < 0.05, **p < 0.01, and ***p < 0.001.

μ100 μ m

Supplemental Figure 8. Protective effects of IP-vaccination are TGFβ dependent. Animals were vaccinated with STAT6-IP or CP. During sensitization some animals were treated intranasally with a pan neutralizing anti-TGFβ monoclonal antibody (αTGFβ) as described in the methods. Following challenge animals lungs were prepared and stained with H/E as described in the methods. αTGFβ treatment (IP+αTGFβ) was sufficient to restore allergic sensitization to the levels of unvaccinated allergic mice (OVA). Magnification 100×.

IP-Isotype CP β

TGF-α

CP-CP-Is

otype0

200

400

600

800

Num

ber o

f cel

ls p

er fi

eld

IP-Isotype CP β

TGF-α

CP-CP-Is

otype0

100

200

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IL4

(pg/

ml)

IP-Isotyp

e CP β

TGF-α

CP-CP-Is

otype

0

1000

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4000

IL13

(pg/

ml)

IP-Isotyp

e CP β

TGF-α

CP-CP-Is

otype

0

200

400

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1000

IL10

(pg/

ml)

IP-Isotype CP β

TGF-α

CP-CP-Is

otype0

100

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500

OVA

-Spe

cific-

IgE

(ng/

ml)

IP-Isotype CP β

TGF-α

CP-CP-Is

otype0

50

100

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250

TGFβ

(pg/

ml)

IP-Isotype CP β

TGF-α

CP + CP-Is

otype0

5

10

15

Mea

n Fl

ores

cenc

e In

tens

ity

Baseline 3.125 25 500

10

20

30

40

Methacholine (mg/ml)R

esis

tanc

e (R

max

, cm

H2O

.s/m

l)

Baseline 3.125 25 500

10

20

30

40

50

Methacholine (mg/ml)

Resi

stan

ce (R

max

, cm

H2O

.s/m

l)

a b c d

e fg

h i j

Even

ts

IL4

CP + αTGF-β

CPIP + Isotype

CP + Isotype

Supplemental Figure 9. Pan neutralizing anti-TGFβ monoclonal antibody (αTGFβ) has no effect in STAT6-CP vaccinated mice while isotype control antibody treatment has no effect in STAT6-IP vaccinated animals. (a) Differential cell counts were obtained from recovered BAL fluid and stained with Diff-Quick. White columns denote macrophages (□) lymphocytes ( ) light grey, neutrophils ( ) dark grey and eosinophils (■) black. IP-vaccinated animals treated with isotype control antibody are protected from airways inflammation while CP-vaccinated animals show increased inflammatory cells in BAL, unaffected by either αTGFβ or isotype antibody treatment. (b OVA-specific-IgE was determined in serum samples by ELISA. (c-f) Splenocytes from IP, CP vaccinated, sensitized with or without αTGFβ or isotype control were cultured in triplicate in the presence of OVA for and IL4 (c), IL10 (d), IL13 (e) and TGFβ (f) were determined from supernatants as described in Methods. (g, h) αTGFβ or isotype control treatment during sensitization has no effect on frequency or mean fluorescence intensity of IL4+ T cells assessed by flow cytometry. (i,j) Animals were treated with αTGFβor isotype control antibodies during allergen sensitization (i) or challenge (j) and bronchial airway hyperresponsiveness to methacholine was assessed using the Flexivent small animal ventilator. IP + isotype (●), CP (■), CP + αTGFβ (▲) and CP + isotype (▼), respectively.

PBS βTGF-α

PBS + OVA β

TGF-α

OVA +

IP βTGF-α

IP +

0

200

400

600

IL4

(pg/

ml)

***

PBS βTGF-α

PBS + OVA β

TGF-α

OVA +

IP β

TGF-α

IP +

0

500

1000

1500

IL10

(pg/

ml)

***

PBS βTGFα

PBS + OVA β

TGFα

OVA +

IP βTGFα

IP +

0

1000

2000

3000

4000

IL13

(pg/

ml)

***

PBS β

TGFα

PBS + OVA β

TGFα

OVA +

IP β

TGFα

IP +

0

1000

2000

3000

4000

IFN

γ (pg

/ml)

***

PBS β

TGFα

PBS + OVA β

TGFα

OVA +

IP β

TGFα

IP +

0

50

100

150

200

250

TGFβ

(pg/

ml)

***

a b

c d

e

*** ***

*** ***

***

Supplemental Figure 10. Pan neutralizing anti-TGFβ monoclonal antibody (αTGFβ) blocked the immunomodulatory effects of STAT-6-IP vaccination during sensitization. Splenocytes, from IP or CP vaccinated animals sensitized with OVA, with and without αTGFβ treatment, were cultured in the presence of OVA and IL4 (a), IL10 (b), IL13 (c), INFγ (d) and TGFβ (e) were determined from supernatants. αTGFβ treatment restored the allergic cytokine profiles in vaccinated mouse splenocytes. ***p < 0.001