Science - USA (2019-02-15)

in the absence of stimulation and faster dif-
ferentiation into CD138+plasma cells upon lipo-
polysaccharide (LPS) stimulation ( 25 , 26 ). In
addition, unlike B2 cells, they are refractory to
anti-IgM F(ab) 2 – induced BCR engagement and
show efficient class switching to IgA upon stim-
ulation with B cell–activating factor (BAFF),
LPS, and transforming growth factor–b(TGF-b)

( 27 – 29 ). Without exception, the response of peri-

toneal B2→B1 cells to these stimuli was similar

to that of B1 cells (Fig. 3E and fig. S10). Thus,

B2→B1 cells not only acquired the B1 cell surface

phenotype, but also adopted B1-typical functional

properties in vitro and in vivo.

To expand the analysis of the identity of

B2→B1 cells beyond the limited number of sur-

face markers, we performed RNA sequencing

(RNA-seq) of peritoneal B2→B1 cells and com-

pared their transcriptome to that of peritoneal

B1 and splenic B2 control cells. In terms of a

previously defined B1 signature consisting of 14

B1-specific and 14 B2-specific genes ( 30 ), B2→B1

cells showed an expression profile almost iden-

tical to that of B1 control cells (Fig. 4A). This

Grafet al.,Science 363 , 748–753 (2019) 15 February 2019 4of6

E

α-IgM

LPS

LPS

BAFF

TGFβ

FSC

PI

CD19

CD138

IgM

IgA

IgM

IgG2a/2b

B2 ctrl cells

(wild-type)

B1 ctrl cells

(B1-8VH12)

B2→B1 cells

by TAT-Cre by CreERT2

A

5C5 (B1)

Ac146 (B2)

5C5 (B1)

IgMa

Spleen Peritoneal cavity B

%B2

→

B1 of IgM

a B cells

%IgM

a of B cells

5C5 (B1)

IgMa

C Pleural cavity

D

84.6

B2→B1 B2→B1

B2→B1

SP PeC

IgMa+ IgMa+

14.9 83.7

88.0 99.7

Ac146+

10.6

Ac146+

0.17

Live Live Live Live

IgG2a/2b+ IgG2a/2b+ IgG2a/2b+ IgG2a/2b+

****

0

20

40

60

80

100

0

50

100

SP PeC

****

1:80 1:320 1:1280 1:5120

0

0.4

0.8

1.2

B1-mouse

B2→B1

Bl6

Rag2-/-

Antibody titer (OD 405)

Serum dilution

5C5-binding IgMa Abs

Fig. 3. B2→B1 cells acquire the functional
characteristics of B1 cells.(A) Flow
cytometric analysis of the experimental
IgMaB cells (by TAT-Cre) within all B cells in
recipients 30 days after transfer in the
spleen (SP) and peritoneal cavity (PeC) (top)
and the 5C5+B2→B1 cells within these cells
(bottom). (B) Quantification of the data
shown in (A). Each dot represents one
recipient mouse analyzed in one of five
independent experiments. ****P< 0.0001
(pairedttest). (C) Flow cytometry showing
the B2→B1 cells within the B cells in the
pleural cavity. (D) Enzyme-linked immuno-
sorbent assay measurements of 5C5-specific
antibodies with IgMaallotype in the serum
of three B2→B1 recipients (30 days after
transfer) and the indicated controls (P<0.001
between B2→B1 recipients and negative
controls). OD 405, optical density at
wavelength 405 nm. (E) Stimulation of
wild-type splenic B2 cells, control peritoneal
cavity 5C5+B1 cells (from B1 mice), and
peritoneal B2→B1 cells isolated 30 days after
transfer. The BCR switch was induced by
TAT-Cre or CreERT2/tamoxifen, as indicated.
Cells were stimulated by anti-IgM for 4 days
(first row), LPS for 2 days (second row), or
BAFF,LPS,andTGF-bfor 4 days (last two
rows) and analyzed by flow cytometry. Gates
show survival and activation of cells based on
propidium iodide (PI) and size (first row),
plasma cell differentiation based on CD138
(secondrow),andisotypeswitchingfromIgM
to IgA or IgG2a/2b (last two rows, gated on
IgM-negative cells). Data are representative of
two independent experiments.

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