Science - USA (2022-02-11)

fluorescence-minus-one (FMO) controls cor-
respond to cells that were stained with mAbs
for the detection of lineage markers but not
for the indicated molecule. Cell surface C3 was
detected using biotinylated anti-C3 mAbs clone
11H9 (Novus Biological), clone 3d29 [kindly
provided by V. M. Holers and J. M. Thurman,
School of Medicine, University of Colorado,
Denver, Anschutz Medical Campus ( 32 )], or

polyclonal Abs (pAbs) HP8012 (Hycult) and

55500 (MP Biomedical) (table S4) with sub-

sequentincubationwithBV605-,APC-,or

APCCy7-conjugated streptavidin. All FMO

controls for C3 surface staining correspond

to cells that were incubated with mAbs for

the detection of lineage markers, including

streptavidin-BV605/APC-Cy7/APC but no bio-

tinylated anti-C3 Abs. All staining steps were

performed on ice (30 min) and isotype-matched

control Abs were used where required. Cells were

analyzed using a LSRFortessa flow cytometer (BD

Biosciences) in the Melbourne Cytometry

Platform (University of Melbourne) and FlowJo

software (Tree Star) with exclusion of cell doublets

and dead cells, in all cases identified on the basis

of forward and side scatter (FSC and SSC)

and propidium iodide (PI) staining (fig. S2).

Schrieket al.,Science 375 , eabf7470 (2022) 11 February 2022 9 of 12

Fig. 8. MZ B cells present pMHC II complexes
trogocytosed from cDC1s in vivo.(Aand
B) Representative flow cytometry histograms
(left) and bar graphs with MFI values (right)
of C3 surface expression on wild-type or
Cr2–/–total, FO, or MZ B cells of mixed-BM
chimera mice (wild-type orC3–/–recipients),
reconstituted with a 1:1 mix of wild-type
andCr2–/–BM (A) or on FO and MZ B cells
of the indicated wild-type or mutant mice
(B). Bar graphs display data pooled from
two independent experiments, with each
symbol representing an individual mouse
(n= 4 or 5 per experiment); bars denote
mean ± SD. P<0.002,*P< 0.0002,
**P< 0.0001 [Welch’s ANOVA test
(no assumption of equal variances) followed
by pairwise comparison (A) or by Games-Howell
multiple-comparisons test (B), adjusted
Pvalue (95% CI)]. (C) Cartoon representing
the capture of anti-Clec9A mAb fused to
the I-E peptide (I-Ea46-72) by cDC1s, followed
by intracellular processing and presentation of
I-E peptide by MHC II (I-Ab) and detection of
the complex on the cell surface with YAe mAb.
(D) Left: Surface expression of I-Ab+ IEpep
(YAe) on splenic cDC1s, cDC2s, total B cells,
and MZ B cells of the indicated wild-type
or mutant mice immunized with anti–Clec9A-
IEpep or control isotype-IEpep mAb. Right:
Bar graphs with MFI values of YAe surface
expression; data pooled from two independent
experiments, with each symbol representing an
individual mouse (n= 2 or 3 per experiment),
displayed as means ± SD. *P< 0.0332, P<
0.002, *P< 0.0002, *P< 0.0001 [Welch’s
ANOVA test (no assumption of equal variances)
followed by pairwise comparison within each
group (cell type),Pvalue (all 95% CI)].
(E) Proliferation of OT-II cells incubated with
increasing numbers of sort-purified FO or
MZ B cells from the indicated wild-type or
mutant mice immunized with anti-Clec9A mAb
conjugated with OVA. Left: Dividing OT-II
cells, gated as shown in the representative flow
cytometry plots. Right: Numbers of proliferated
OT-II cells pooled from two independent
experiments, with each symbol representing
a technical replicate (n= 3 per experiment),
displayed as means ± SD. P< 0.0332,
P< 0.0001 (one-way ANOVA followed by
Bonferroni’s multiple-comparisons test).

A

0

1000

2000

3000

4000

0

2500

5000

7500

10000

C3

% of max.

WT

recipient

C3−/−

recipient

all B cell FO B cell MZ B cell

B

FMO WT (Ly5.1+Ly5.2−) Cr2−/−(Ly5.1−Ly5.2+)

****ns

****

***

0

1000

2000

3000

4000

Cr2

WT BM−/−^ BM

WT

(recipient)

C3−/−

(recipient)

Cr2

WT BM−/−^ BM

****ns

**

***

****ns

****ns

all B cell FO B cell MZ B cell

C3 (gMFI over FMO)

FMO

WT

MHC IIKRKI/KI

0

500

1000

1500

2000

2500

3000

0

1000

2000

3000

4000

(^100005000)
15000
20000
H2-Aa−/−
FO B cell MZ B cell
C3
% of max.
ΜΗC IIKRKI/KI
×Cr2−/−
Cr2−/−
C3 (gMFI over FMO)
FO B cell MZ B cell

---

---

---

**
Cr2
WT BM−/−^ BM
WT
(recipient)
C3−/−
(recipient)
Cr2
WT BM−/−^ BM
Cr2
WT BM−/− BM
WT
(recipient)
C3−/−
(recipient)
Cr2
WT BM−/−^ BM
ns

---

**

---

WT
ΜΗC
IIK
RKI
/KI
ΜΗC
IIK
RK
I/KI×Cr
2 −/
−
H2-A
a−/
−
Cr2
−/−
C
D
FMO isotype ctr. WT MHC IIKRKI/KI MHC IIKRKI/KI×C3−/−
cDC1 cDC2 all B cell MZ B cell
YAe (IEpep in MHC II)
% of max.
WT
MHC IIKRKI/KI
MHC IIKRKI/KI
×C3−/−
E
0.69
4.62
0.47
CTV
CD4
OT-II
WT
MHC IIKRKI/KI
MHC IIKRKI/KI
×C3−/−^0 100 200 300 400
0
300
600
900
1200
1500
1800
number of proliferated OT-II

---

ns
ns
0
1000
2000
3000
4000
5000
6000
Number (× 103 ) of FO/MZ B cells
WT MHC IIKRKI/KI MHC IIKRKI/KI MHC IIKRKI/KI×C3−/−
all
B cell
MZ
B cell
cDC1 cDC2
WT MHC IIKRKI/KI MHC IIKRKI/KI×C3−/−
YAe (IEpep in MHC II) over isotype ctrl.

---

---

**
ns
ns
ns ns

• 
  


• 
  

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• 
  

  0.56
  1.12
  1.12
  FO B cell MZ B cell
  FO B cell MZ B cell
  0 100 200 300 400
  0
  300
  600
  900
  1200
  1500
  1800
  ns *
  ns
  WT
  ΜΗC
  IIKR
  KI/KI
  ΜΗC IIK
  RKI/K
  I×Cr2
  −/−
  H2-Aa
  −/−
  Cr2
  −/−
  RESEARCH | RESEARCH ARTICLE