Science - USA (2022-02-11)

enriched by density gradient centrifugation
(at 2237g) with 1.077 g/cm^3 Nycodenz (Axis
shield) and subsequent negative depletion
using rat mAbs against CD3 (KT3-1.1), Thy1
(T24/31.7), Ly-76 (Ter119), B220 (RA3-6B2),
and Ly-6C/G (RB6-8C5) and BioMag anti-rat
IgG-coupled magnetic beads (20ml/10^6 cells,
Qiagen), resulting in 75 to 90% purity. Similarly,
cDCs from total lymph nodes were enriched

through enzymatic digestion [0.1% DNase I

and collagenase type III (1 mg/ml)] and Nyco-

denz (1.077 g/cm^3 , Axis shield) gradient cen-

trifugation (at 2237g). Splenic B cells were

isolated from whole-splenocyte suspensions

through gradient centrifugation (at 2237g)

with Ficoll-Paque Plus (GE Healthcare) and

subsequent negative depletion using FITC-

conjugated mAb against CD4 (GK1.5, 1.6mg/

ml), Ly76 (TER119, 1.6mg/ml), and CD43 (S7,

1.6mg/ml) and magnetic anti-FITC MicroBeads

(2ml/10^6 cells, Miltenyi Biotec), resulting in 95

to 98% purity.

For flow cytometry analysis, purified murine

cells were washed in EDTA-BSS with 2% (v/v)

FCS, Fc receptorÐblocked (1:50, Miltenyi

Biotec), and incubated with mAb for the

detection of surface markers (table S4). All

Schrieket al.,Science 375 , eabf7470 (2022) 11 February 2022 8 of 12

Fig. 7. B cell trogocytosis of cDC
membrane is MHC IIÐ, C3-, and CR2-
dependent.(A) Trogocytic acquisition of CD8
from cDCs by wild-type orCr2–/–B cells after
their incubation with wild-type or mutant
cDCs as indicated, with bar graphs (right)
representing the percentage of CD8+B cells,
determined as shown in the flow cytometry
plots (left). Each data point represents
the measurement of a technical replicate (n=
3), displayed as mean ± SD. *P< 0.0332,
*P< 0.0002, ***P< 0.0001 [Welch’s
ANOVA test (no assumption of equal varian-
ces) followed by Games-Howell multiple-
comparisons test, adjustedPvalue (95%
CI)]. (B) Representative B220 versus CD8
and CD11c plots of flow cytometry analysis of
low-density splenocytes from the indicated
wild-type or mutant mice, showing the
proportion of trogocytic (CD8+and CD11c+)
B cells. Plots are representative of at least
two independent experiments with two
or three individual mice per experiment.
(C) Frequency of trogocytic MZ B cells
among low-density splenocytes of wild-type
orC3–/–mice. (D) Number of splenic cDC1s
and cDC2s in the indicated wild-type or
mutant mice. (E) Number of cDC1s and
cDC2s (left) and frequency of trogocytic
B cells (right) identified as shown in (B) in
the indicated wild-type or mutant mice.
(F) Number of wild-type orMarch1–/–trogocytic
B cells, cDC1s, and cDC2s present in the
spleens of mixed-BM chimeras (wild-type or
C3–/–recipient mice), reconstituted with a
1:1 mix of wild-type andMarch1–/–BM.
Graphs in (C) to (F) display data pooled from
six (C) or two [(D) to (F)] independent
experiments, with each symbol representing
an individual mouse (n= 2 to 5 per experi-
ment); bars denote mean ± SD. P< 0.0332,
P< 0.002, *P< 0.0002, **P< 0.0001
[independent-samplesttest with Welch’s
correction (no assumption of equal variances)
in (C), Welch’s ANOVA test (no assumption of
equal variances) followed by Games-Howell
multiple-comparisons test in (D) and (E),
or followed by pairwise comparison in (F),
two-tailed (C), or adjusted [(D) to (F)]
Pvalue (all 95% CI)].

B

C

11.7

2.30

12.2

1.41

20.1

1.53

3.35

16.5

0.91

1.74 1.79

2.08

B220

CD8

B220

CD11c

CD8+ B cell CD11c+ B cell

WT

March1−/−

March1−/−

×H2-Aa−/−

ΜΗC IIKRKI/KI

ΜΗC IIKRKI/KI

×C3−/−

March1−/−

×C3−/−

D

% of MZ B c

ells

CD8+

MZ B cell

CD11c+

MZ B cell

E

F

0

15

30

45

60

0

30

60

90

120

150

0

3

6

9

12

% of live

0

2

4

6

8

10

ns ns ns ns

** ** *** ***

WT

ΜΗC

IIKR

KI/KI×

Cr2

−/−

ΜΗC IIKR

KI/KI WT

ΜΗC

IIK

RKI/K

I×Cr2

−/−

ΜΗC IIK

RKI/K

I

WT

ΜΗC

IIK

RKI/KI

×Cr2

−/−

ΜΗC

IIK

RKI/KI WT

ΜΗC IIKR

KI/KI×

Cr2

−/−

ΜΗC IIKR

KI/KI

cDC1 cDC2 CD8+ B cell CD11c+ B cell

number

(×10

4 )

0

7

14

21

****

****ns

0

5

10

15

20

****

ns****ns

0

10

20

30

40

(^50) **

---

ns
0
30
60
90
120
150
180

---

**
ns
0
9
18
27
36
45
54
ns
ns
0
40
80
120
160
200
cDC1

---

---

ns
ns
cDC2

---

---

number
(×10
4 )
WT
ΜΗC IIKR
KI/KI×
C^3
−/−
ΜΗC IIKR
KI/KI
March1
−/−×
C^3
−/−
March1
−/−
WT
ΜΗC IIKR
KI/KI×
C3
−/−
ΜΗC IIK
RK
I/KI
March1
−/−×
C^3
−/−
March1
−/−
number
(×10
4 )
0
2
4
6
8
0
5
10
15
20
WT C^3
−/−
WTC3
−/−

---

low-density splenocytes
March1
WT BM−/−^ BM
March1
WT BM−/− BM
March1
WT BM−/− BM
March1
WT BM−/− BM
cDC1 cDC2
March1
WT BM−/− BM
March1
WT BM−/− BM
March1
WT BM−/− BM
March1
WT BM−/− BM
WT
(recipient)
C3−/−
(recipient)
WT
(recipient)
C3−/−
(recipient)
WT
(recipient)
C3−/−
(recipient)
WT
(recipient)
C3−/−
(recipient)
CD8+
B cell
CD11c+
B cell
number
(×1
(^40)
)
B220
CD8
WT
B cell
Cr2−/−
B cell
CD8
WT
cDC
no
cDC
MHC IIKRKI/KI
cDC
MHC IIKRKI/KI×C3−/−
cDC
0.58
0.34
12.8 38.9 7.84
13.6 11.1 7.33
0
10
20
30
40
50
0
10
20
30
40
50
% of B cells
WT B Cell Cr2−/−B Cell
CD8+ B cell

---

---

• 
  

  ---

  
  

  WT
  no cDC cDC
  ΜΗC IIK
  RK
  I/KI×C3
  −/− cDC
  ΜΗC IIKR
  KI/KI cDC no cDCWT cDC
  ΜΗC IIKR
  KI/KI×
  C3
  −/− cDC
  ΜΗC IIK
  RK
  I/KI cDC
  A
  ns
  RESEARCH | RESEARCH ARTICLE