binding to MHC II has been conserved through
evolution.
CR2 drives B cell trogocytosis of cDC
membranes containing MHC IIÐC3 complexes
Analysis in vitro confirmed that high surface
MHC II–C3 expression on MHC IIKRKI/KIcDCs
was necessary and sufficient to induce B cell
trogocytosis (Fig. 7A and fig. S6A). C3dg is the
ligand for complement receptor 2 (CR2, also
known as CD21), which is expressed only by
B cells ( 5 ). CR2-deficient B cells lacked the
capacitytotrogocytosemoremembranefrom
C3-decorated cDCs than from wild-type cDCs
(Fig. 7A and fig. S6A). Furthermore, MZ B cells
express higher levels of CR2 than FO B cells(Fig. 2F), and virtually all MZ B cells trogocy-
tosed cDC membrane containing MHC II–C3dg
complexes (fig. S6B).
We next tested the validity of the con-
clusions from our in vitro analyses in vivo.
March1–/–and MHC IIKRKI/KImice accumu-
lated trogocytic (CD8+CD11c+) B cells in their
spleens in a MHC II–and C3-dependent
manner (Fig. 7B). Wild-type spleens contained
a small number of CD8+CD11c+MZ B cells, but
these were absent fromC3–/–spleens (Fig. 7C
and fig. S6C); hence, trogocytosis occurred
constitutively in wild-type mice. Although
lymph node cDCs also displayed MHC II–C3
complexes (Fig. 4B), few trogocytic B cells
were detected in lymph nodes (fig. S6D), as
expected because mouse lymph nodes lack
MZ B cells ( 8 ). The number of splenic cDCs
decreased in all mice where cDCs had enriched
C3 surface expression (Fig. 7D) and B cells
expressed CR2 (Fig. 7E), in lockstep with the
increase in trogocytic MZ B cells. The spleens
of mice deficient in C3 did not contain more
cDCs than those of wild-type mice (fig. S6E);
this finding suggests that the limited amount
of trogocytosis occurring in wild-type mice is
insufficient to affect cDC homeostasis.
To further assess whether cDC number re-
duction and trogocytosis were directly correlated,
we produced mixed-BM chimeras where wild-
type orC3–/–recipient mice were reconstituted
with 1:1 wild-type andMarch1–/–BM. In
wild-type recipient mice, both wild-type and
March1–/–B cells displayed higher trogocytic
activity, but onlyMarch1–/–cDCs were re-
duced in numbers. Neither of these events were
observed in C3-deficient recipients (Fig. 7F).
Thus, the loss of cDCs required the accumula-
tion of MHC II–C3 complexes but was caused by
CR2-dependent MZ B cell trogocytosis (fig. S7).Trogocytic MZ B cells present pMHC II
generated by cDCs
Because the primary mediator of trogocytic
cDC membrane transfer to B cells was CR2
recognition of MHC II–C3dg complexes, we
wondered whether the complexes found on
splenic B cells (Fig. 4B) were in fact acquired
from cDCs. In mixed-BM chimeras where
wild-type recipient mice were reconstituted
with a 1:1 mix of wild-type andCr2–/–BM, all
B cells, and in particular MZ B cells, displayed
more C3 if they expressed CR2 than if they did
not (Fig. 8A). This suggested that although
some of the MHC II–C3 complexes displayed
by B cells probably formed on the B cells
themselves, most were acquired from cDCs.
This is supported by the absence of C3 de-
tection on B cells of mice where MHC II–C3
complexes could not be generated (i.e.,H2-Aa–/–)
or lacked CR2 expression (Fig. 8B), which indi-
cates that virtually all C3 (bound to MHC II)
on the B cell surface was acquired by trogocy-
tosis of cDCs. These results also confirmed thatSchrieket al.,Science 375 , eabf7470 (2022) 11 February 2022 5 of 12
AWT
March1−/−FMOMarch1−/−×C3−/−C3−/−cDC1C3% of max.cDC2Surface C3 cDC1BcDC1cDC1CC3% of max.C3−/−
(recipient)WT+March1−/−BMcDC1 cDC2WT
(recipient)WT+March1−/−BMFMO WT (Ly5.1+Ly5.2−) March1−/−(Ly5.1−Ly5.2+)08001600240032004000030060090012001500
nsns****
ns****
*****
****cDC1 cDC2March1WT BM−/− BM
March1WT BM−/− BMC3 (gMFI over FMO)March1WT BM−/− BM
March1WT BM−/− BMC3−/−
(recipient)WT
(recipient)cDC1 cDC2 pDC mac B cell T cellspleentotal LNneutrophil eosinophilthymusC3 FMO WT March1−/−% of max.C3−/−
(recipient)WT
(recipient)0500010000150002000025000March1WT−/−
C3−/−March1 −
/−×C3−/−
0200040006000800010000March1WT−/−
C3−/−March1 −
/−×C
3 −
/−C3 (gMFI over FMO)******
**
******
**cDC2Fig. 4. Complement C3 deposition on the surface of splenic cDCs.(A) Representative flow cytometry
histograms (left) and bar graphs with MFI values (right) of C3 surface expression on splenic cDC1s and
cDC2s purified from the indicated mice. (B) Representative histograms of flow cytometry analysis of C3
surface levels on the indicated cell types in spleen, lymph nodes (LN), and thymus of wild-type orMarch1–/–
mice. Histograms are representative of at least two independent experiments with two or three individual
mice per experiment. (C) Representative flow cytometry histograms (left) and bar graphs with MFI values
(right) of C3 surface expression on wild-type andMarch1–/–cDC1s and cDC2s from mixed-BM chimeras
where wild-type orC3–/–recipient mice were reconstituted with a 1:1 mix of wild-type andMarch1–/–BM.
Graphs in (A) and (C) display data pooled from a minimum of two independent experiments, with each
symbol representing an individual mouse (n= 3 to 5 per experiment); bars denote mean ± SD. *P< 0.0332,
P< 0.002, **P< 0.0001 [WelchÕs ANOVA test (no assumption of equal variances) followed by Games-
Howell multiple-comparisons test (A) or by pairwise comparison (C), adjustedPvalue (95% CI)].
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