Science - USA (2022-02-11)

Liuet al.,Science 375 , eabi5965 (2022) 11 February 2022 2 of 13

A

DCIR2

CD8

12.6

74.0

25.2

46.4

19.7

41.8

43.8

28.5

Control

Gna13cKO

Arhgef1

Arhgef1 Gna13cKO

100

80

60

40

20

0

cDC2 % in DCs

****

****

****

0.2

0

dKO

****

****

****

52.0 47.2

CD45.1

B220

CD45.1

CD11c

43.1 54.6 43.2 55.3

B220+ B220I-AbhiCD11chi DCIR2+ DC

59.0 40.2

21.9 76.0 17.0 82.4

Competitive competency

=

CD45.1^ / CD45.1+ target pop

CD45.1 / CD45.1+ comparison pop

1.0

0.8

0.6

0.4

0.2

0

Competency

****

cDC2/FO

CD45.2 PE

CD11c

80

60

40

20

0

n.s.

*

Test

W T:

Gna13

cKO

W

T:Gna13

WT

80

60

40

20

0

n.s. ***

PE

+ % in cDC2

Control

100 0.04

0.03

0.02

0.01

0

cDC2 % in WBCs

Gna13

cKO

Gna13

WT

*

Control

0.4

0.6

0.8

1.0

cDC2 % in splenocytes

C Control

Gna13cKO

Arhgef1

Arhgef1 Gna13cKO

Gna13 IgD DCIR2

cKO

Arhgef1

J

B

DE

F

H

PE

+ % in cDC2

0.04

0.03

0.02

0.01

0

cDC2 % in WBCs

**

Arhgef1Arhgef1

Pre-gated on B220CD11chiI-Abhi

W T:

Arhgef1

W T:

Arhgef1

G

3

2

1

0

Competency

cDC2/FO

****

WT

:Gna13

cKO

W

T:Gna13

WT

45.5 50.7

CD8+ DC

23.7 71.9

WT :

Arhgef1

WT :

Arhgef1

WT

42.1 42.4

Gna13WT

Control Test

43.8

WT Gna13cKO

35.4

I

WT :

Gna13

cKO

WT :

Gna13

WT

W T:

Arhgef1

W T:

Arhgef1

Fig. 1. Ga 13 -ArhGEF1 signaling pathway is required in splenic cDC2s.(Aand
B) Representative (A) flow cytometry profiles and (B) frequencies of DCIR2+CD8−
cDC2s in (top) total B220−CD11chiI-AbhiDCs and (bottom) total splenocytes inArhgef1−/−,
Cd11c-creGna13fl/fl(labeled asGna13cKO),Arhgef1−/−Cd11c-creGna13fl/fl(labeled as
Arhgef1−/−Gna13cKOor dKO), and control mice. Data are pooled from three
independent experiments. (C) Representative distribution patterns of DCIR2+cDC2s
(red) relative to B cells [immunoglobulin D (IgD), blue] in spleens of mice of the
indicated genotypes. Scale bar, 200mm. Sections are representative of multiple cross
sections from at least three mice of each type. (DtoF) Mixed (50:50) BM chimeras
were made with CD45.1 WT (Arhgef1+/+) and CD45.2Arhgef1+/−orArhgef1−/−BM
cells. (D) Representative flow cytometry profiles show gating strategies. (E) Equation

for calculating the competitive competencies of CD45.2+(gated as CD45.1−)

population. (F) Plots showing CD45.2+competency values in individual chimeras for

the cDC2 compartment compared with B220+follicular (FO) B cells. (G) Plots showing

CD45.2+competency values in WT:Gna13WTand WT:Gna13cKOchimeras for the

cDC2 compartment compared with B220+follicular (FO) B cells. (H)Flowcytometry

profiles and (I) frequencies of in vivo anti-CD45-PEÐlabeled cDC2s of indicated

genotyped cells in (left) WT:Arhgef1−/−or (right) WT:Gna13cKOand their control mixed

BM chimeras. Lines connect data from the same animals. (J) Frequencies of cDC2s

in blood of (left)Arhgef1−/−or (right)Gna13cKOand their control mice. In (D) to (J), data

are pooled from two independent experiments. Each symbol represents one mouse,

and lines denote means. *P< 0.05; **P< 0.01; ***P<0.001;****P< 0.0001.

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