Science - USA (2019-02-15)

Grafet al.,Science 363 , 748–753 (2019) 15 February 2019 3of6

A

Day 8

Day 30

Day

4

B2→B1

Spleen 3-8 6-8

5C5+

B2→B1

Ac146+

B2

IgMb

Carriers

28-30

0

100

200

300

400

77

0

500

1000

1500

2000

2500

13

0

100

200

300

400

50

0

500

1000

1500

7

0

50

100

150

200

250

3

0

100

200

300

400

4

0

1000

2000

3000

9

0

50

100

150

200

250

2

0

200

400

600

800

6

BrdU

Time of BrdU pulse (days post transfer)

Ctrl B2 SP B2→B1 PeC (day 30) Ctrl 5C5+ B1a PeC

anti-IL7R

treatment

Isolate mature

B cells

TAT-Cre

treatment

(switch

of BCR)

i.v. transfer with

IgMb carrier B cells

Rag2-/-

Cγ-/-

Analysis

2 weeks 1 day day 4, 8, 30 by FACS

B2

B1

in vitro

B C

D

E

5C5 (B1 cell)

Ac146 (B2 cell)

49

47

99

1

99

1

80

19

53

42

98

1

31

53

97

3

98

2

B1→B2

Spleen

B2→B1

PeC

FSC CD19 CD23 IgD CD43 B220 CD5

B2→B1 SP (day 30)

600

700

800

900

1000

1100

FS

C

102

103

104

105

IgD (mfi)

101

102

103

104

CD43 (mfi)

102

103

104

105

B220 (mfi)

102

103

104

105

CD23 (mfi)

102

103

104

CD19 (mfi)

102

103

104

105

CD5 (mfi

)

Ctrl B2 SPB2→

B1 SP

B2

→

B1 PeC

Ctrl B1a PeC

Ctrl B2 SPB2→

B1 SP

B2

→

B1

PeC

Ctrl B1a PeC

Ctrl B2 SPB2→

B1 SP

B2

→

B1

PeC

Ctrl B1a PeC

Ctrl B2 SPB2→

B1

SP

B2

→

B1 PeC

Ctrl B1a PeC

B2→B1 by TAT-Cre

B2→B1 by CreERT2

****

****

n.s. ****

n.s.

***

****

n.s.

****

n.s.

****

n.s.

n.s.

***

**

Fig. 2. B2→B1 cells acquire the surface phenotype of B1 cells and persist
in vivo after a phase of proliferative expansion.(A) Experimental
scheme: B1 and B2 mice were treated for 2 weeks with antibodies to IL-7R to
block B cell development. Splenic B cells were then treated with TAT-Cre in
vitro and transferred intravenously to immunodeficient recipients the next
day. FACS, fluorescence-activated cell sorting. (B) BCR expression via flow
cytometry on experimental IgMaB cells isolated from the indicated
anatomical sites at the indicated time points after transfer. (C) Proliferation
analysis based on bromodeoxyuridine (BrdU). Animals were fed with BrdU for
the indicated time windows. The BrdU content of the indicated cells was
analyzed on day 8 (left and center) or day 30 (right) by flow cytometry.
(D) Flow cytometric analysis of cell surface phenotype of peritoneal B2→B1

cells (red) isolated from the recipients 30 days after transfer, and of

control splenic B2 (gray) and peritoneal (5C5+) B1a cells (blue) isolated

from wild-type animals. Splenic B2→B1 cells (dashed red curves) are only

shown for the markers in which they differ relative to peritoneal B2→B1 cells

[see (E)]. (E) Summary of the B2→B1 cell surface phenotype from five

independent experiments. Each dot represents the median fluorescence

intensity (mfi) of the indicated surface marker and cell population of one

recipient mouse or control mouse, normalized to the median mfi in PeC

B2→B1 cells (set to 1000) per experiment. Tamoxifen/CreERT2-derived

B2→B1 cells are colored turquoise (see fig. S7 for details). ****P<0.0001,

***P< 0.001, **P< 0.01 (ordinary one-way analysis of variance and

Bonferroni multiple-comparisons test); n.s., not significant.

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