Science - USA (2022-02-25)

SCIENCEscience.org 25 FEBRUARY 2022¥VOL 375 ISSUE 6583 861

Fig. 2. Tr-ILC3s show long-term
persistence.(A) Experimental
design for (B) to (H).
RorcGFP×Il22TdTandNcr1GFP×Il22TdT
mice received ciprofloxacin
(Abx; 100 mg kg–^1 day–^1 ) after
C. rodentiuminfection. Four months
later, the mice were reinfected
withC. rodentium(CRACR).
(B) FecalC. rodentiumcounts
(CR,n= 8; CRACR,n= 12). CFU,
colony forming units. (Cand
D) Absolute numbers of total (C)
(n= 3 to 8) andIl22TdT+(D)
(n= 3 to 9) ILC3s in the small
intestine lamina propria. (E) Abso-
lute numbers of intestinal IL-22+
ILC3s (protein;n= 3 to 5) after
ex vivo IL-23 and IL-1bstimulation
3 days after infection or reinfection.
(FandG) Frequency and absolute
numbers of ILC3s (F) andIl22TdT+ILC3
(G) subsets in CRACR mice were
determined (n= 3 to 5). DN, double
negative. (H) Fate-mapping
protocol.Id2CreERT2+×Rosa26RFP
mice received ciprofloxacin
(Abx; 100 mg kg–^1 day–^1 ) 3 days afterC. rodentiuminfection. Mice received tamoxifen (Tmx) by intraperitoneal injection and were analyzed as shown. (I) Analysis of
Id2RFP+cells in ILC1 (CD3−NKp46+NK1.1+), ILC2 (CD3−CD127+CD90.2+KLRG1+), and ILC3 subsets (left) for the protocol shown in (H). Percentages ofId2RFP+cells of the total
ILCs in CR, CRA, and CRACR mice were determined (n= 3 to 5) (right). Each graph corresponds to the mean ± SEM of the values obtained. ns indicates not significant, *P< 0.05,
and **P< 0.01 using two-tailed Mann-Whitney test.

ARorcGFP Il22TdT

Ncr1GFPIl22TdT

CR Abx

D0 D3 D120

CR

CR CRACR

B C D

CR CRACR

100

101

102

103

104

105

106

107

108

109

CFU/g of Feces

ILC3 (

10

4 )

Time (mo)

**

• +++++

0

25

50

75

100

125

150

*

**

14

CR

---+ - + CRACR

ns

Time (mo)

• +++++
  0

20

40

60

100

(^130) *
**
14
CR
---+ - + CRACR
Il22
TdT+
ILC3 (
10
4 )
ns

• 
  

+

CRACR

4 months

NKp46+CCR6+ DN

0

2

4

6

8

10

12

Il22

TdT+

ILC3 (

10

4 ) **

ns

ns

7.4 38.1 28.1

NKp46+

Il22

(TdT)

CCR6+DN

ILC3 - after reinfection (D3)

4 months after primo infection

NKp46+CCR6+ DN

0

5

10

15

20

25

ILC3 ( 10

4 )

G

• 
  

+

CRACR

4 months

ns

**

*

F

Ncr1

(GFP)

CCR6

ILC3 - after reinfection (D3)

4 months after primo infection

E

CRCRACR

IL-22

+ ILC3 (

10

4 )

ns

H

TmxCR

-D4 D0 D3

Analysis

CR

D0 D4 D8

Abx

Tmx

CR

D30

Analysis

ID2+ RFP+

Tmx

Id2CreERT2+

Rosa26RFP

0

1

2

3

4

5

6

ILC1ILC2ILC3

I

ILC1ILC2ILC3

Id2

(RFP)

ILC1ILC2ILC3 NKp46+DNCCR6+ CR CRA CRACR

CR (D3) CRA (D30) CRACR (D33) CRACR (D33)

(RFP )

0

10

20

30

40

50

60

ILC1

ILC2

ILC3

Id2

RFP+

cells (% of ILC)

25.427.945.5 35.640.9 57.1 5.5 4.446.5 50.951.936.2

Id2

18

(^3841)
Fig. 3. Tr-ILC3s show
enhanced IL-22Ðmediated
protection.(A) Experimen-
tal design for (B) to (E).
Rag2−/−andRag2−/−Il22−/−
mice received ciprofloxacin
(Abx; 100 mg kg–^1 day–^1 )
afterC. rodentiuminfection.
One month later, the mice
were reinfected with CR
(CRACR). (B) Analysis of
ILC3s (left) and IL-22+
ILC3s (right) inRag2−/−
mice (n= 3 to 4). (C) Fecal
C. rodentiumcounts (n= 7).
(D) Survival (left) and body
weight (right) in reinfected
Rag2−/−andRag2−/−IL22−/−
mice (n= 7). (E)C. rodentium
growth monitored by IVIS
imaging 3 days after reinfec-
tion (left). RelativeC. rodentium
growth was determined in
Rag2−/−andRag2−/−IL22−/−
mice (n= 7) (right).
(F) Experimental design for
(G). ILC3s were purified from
infected (CR) or reinfected
(CRACR) mice 3 days after infection and transferred intoC. rodentium–infectedIl22−/−mice. (G) Survival and body weight were assessed at the indicated times after infection [n=4
forIl22−/−andILC3(CR);n=10forIl22−/−and ILC3 (CRACR);n=11forIl22−/−per group, pool of two independent experiments]. Each graph corresponds to the mean ± SEM of the
values obtained. P< 0.05, P< 0.01, and P< 0.001 using Krustal-Wallis test (B), two-tailed Mann-Whitney test [(C) to (G)], or log-rank (Mantel-Cox) test [(D) and (G)].
A
Rag2/
Rag2/Il22/
CR Abx
D0 D3 D30
CR
D40
CR CRACR
BC
0
50
100
150
200
250
300
ILC3 (
10
4 )
0
25
50
75
100
125
150
IL-22

• ILC3 (
  10
  4 )
  103
  104
  105
  106
  107
  108
  109
  CFU/g of Feces

• +++CR
  --- +CRACR
  1 mo

CR

CRACR

1 mo

CR CRACR

Rag2–/– Rag2–/–

• +++
  --- +

*

**

D

40 42 44

0

20

40

60

80

100

Survival (%)

Rag2

Rag2 Il22

***

Time (days)

Rag2

Rag2 Il22

30 33 34 35 37 40 41

-16

-12

-8

-4

0

4

Body weight change (%)

Time (days)

***

***

***

Rag2 (CRACR) Rag2 Il22 (CRACR)

Rag2

Rag2 Il22

E

100

101

102

103

104

105

106

107

Radiance (p/sec/cm

2 /sr)

***

104 105

Radiance (p/sec/cm /sr) Radiance (p/sec/cm /sr)

(^1234) x10
0 3 4 5 7 9 10 11 12 13 16 18
-25
-20
-15
-10
-5
0
5
Time (day)
Control
+ILC3 (CR)
+ILC3 (CRACR)
Il22
0 4 8 12 16 20 24
Time (day)
Cell adoptive
transfer

---

Body weight change (%)
CR Survival (%)
D0 D3 D6 D9
Il22/
FG
ILC3
0
20
40
60
80
100
/ /

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