Science - USA (2019-01-04)

Biosciences.Gata3fl/fl( 37 ),Foxp3YFP-Cre( 38 )and
Il17aCre( 26 ) have been previously described and
were generously provided by J. Zhu (NIAID, NIH),
A. Rudensky (Memorial Sloan Kettering Cancer
Center), and B. Stockinger (Francis Crick Insti-
tute), respectively.Foxp3gfp( 39 ), CD45.1 (B6.SJL-
PtprcaPepcb/BoyJ),Tcra−/−(B6.129S2-Tcratm1Mom/J)
( 40 ), CD45.1Rag1−/−,Il13−/−( 41 ), and CreERT2-
Gata3fl/flmice ( 42 ) were purchased from the

NIAID-Taconic Exchange.Tcra+/−mice were

generated by breedingTcra−/−mice with C57BL/6

WT mice.Foxp3DTR(B6.129(Cg)-Foxp3tm3(DTR/GFP)

Ayr/J) ( 11 )andR26ReYFP(B6.129X1-Gt(ROSA)

26Sortm1(EYFP)Cos/J) ( 43 )micewerepurchased

from The Jackson Laboratory.CD4CreIl18r1fl/fl

andIl18r1fl/flcontrolmicewerekindlyprovided

by G. Trinchieri (NCI, NIH). All mice were bred

and maintained under SPF conditions at an

American Association for the Accreditation of

Laboratory Animal Care (AAALAC)–accredited

animal facility at NIAID and housed in accord-

ance with the procedures outlined in the Guide

for the Care and Use of Laboratory Animals. All

experiments were performed at NIAID under an

Animal Study Proposal (LPD-11E or LPD-68E)

approved by the NIAID Animal Care and Use

Committee. Sex- and age-matched mice between

Harrisonet al.,Science 363 , eaat6280 (2019) 4 January 2019 8of11

i.d. PBS i.d. IL-18

IL-17A

IL-5

29±2 0.4±0.2

0.6±0.4

15±3 0.4±0.1

0.5±0.3

9±3 2±1

15±4

15±4 3±2

12±4

eYFP (

Il17a

FM

)

CCR6

CD8+ T cells

Cytokine

+ (%)

IL-17AFM+^ Tc17

0

10

20

30

PBS IL-18

*

*

*

PBS IL-18

0

10

20

30

Cytokine

+ (%)

*

*

*

IL-17AFM-^ Tc17

IL-17A+

IL-17A+IL-5+

IL-5+

IL-17A+

IL-17A+IL-5+

IL-5+

i.d. PBS i.d. IL-18

IL-17A

IL-5

A

IL-17A+

IL-17A+IL-5+

IL-5+

0

10

20

30

Cytokine

+ (%)

CD4+ CCR6+ IL-17AFM+

**

**

**

PBS IL-18

Cytokine

+ (%)

0

10

20

30

CD4+ CCR6+^ IL17AFM-

**

**

**

PBS IL-18

B

C ILC2

CD4+

Tc17

0 24487296

0

2

4

6

IL-5

+ Cells

(x10

3 )

hours post IL-18 injection

IL-13

+ Cells

(x10

3 )

hours post IL-18 injection

0 24487296

0

1

2

3

Ctrl

0

1

2

3

4

Eosinophils (x10

4 )

**

i.d. PBS

i.d. IL-18

Isotype

anti-IL-5

D E

0

0.5

1.0

1.5

2.0

Epidermal tongue (x 10

3 μm) **

S. epidermidis

Isotype

anti-IL-13

Naive S. epidermidis

WT Bo

wie

Tg +

Epidermal tongue (×10 isotype

3 μm)

0

0.5

1.0

1.5

2.0

WT

Il13-/-

** *** * *

no transfer

WT

Bo

wie

Tg +

anti-IL-13

F

-8 -6 -4 -2^0

Extracellular matrix organization

Striated muscle contraction

Muscle contraction

Degradation of extracellular matrix

Enrichment score

(Log 10 p adjusted)

IL-13-dependent pathways

G

IL-17A+

IL-17A+IL-5+

IL-5+

Fig. 6. Commensal-specific T cell plasticity and IL-13 production
promote wound repair.(A) Representative contour plots for gating
strategy of CCR6 and eYFP (enhanced yellow fluorescent protein)
expression by CD8+T cells from the skin ofS. epidermidis–colonized
Il17aCreR26ReYFP(IL-17AFM) mice after i.d. injection of PBS or IL-18.
Contour plots represent IL-5 and IL-17A production potential of IL-17AFM+TC17 (CD8+CCR6+eYFP+) or IL-17AFM−TC17 (CD8+CCR6+eYFP−) T cells after
i.d. injection of PBS or IL-18. (B) Frequencies of TH17 cells with IL-17A–or IL-5–producing potential from the skin ofS. epidermidis–colonized IL-17AFM
mice after i.d. injection of PBS or IL-18. (C) Absolute cell number of IL-5–and IL-13–producing lymphocyte subsets in the skin ofS. epidermidis–colonized
wild-type mice after i.d. injection of IL-18. Data are means ± SD of five mice per group. (D) Absolute number of eosinophils from the skin ofS. epidermidis–
colonized wild-type mice after i.d. injection with PBS or IL-18, and i.p. injection with anti–IL-5 or isotype control. (EandF) Naïve andS. epidermidis–colonized
wild-type andIl13−/−mice, with or without adoptive transfer of BowieTgCD8+T cells before colonization and isotype or anti–IL-13 antibodies at the time of
wounding, were subjected to back-skin punch biopsy. Epithelial tongue length of wound bed–infiltrating keratinocytes was quantified 5 days after wounding.
(G) Pathway analysis using differentially expressed genes between d3 isotype and d3 anti–IL-13 wounding groups was performed using Enrichr and graphed
according to enrichment score for significant Reactome biological processes. Numbers in representative plots indicate means ± SD. Bar graphs show
means ± SD. Data represent at least two experiments with three to seven mice per group. P<0.05,P< 0.01, P< 0.001 as calculated using one-way [(A),
(B), (D)] or two-way [(E), (F)] ANOVA with Holm-Šidák multiple-comparison test.

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