for antibody production, partially because of
lower levels ofIl4andIl21,aswellassurface
Cd154(CD40L) (Fig. 2, C to E, and fig. S5, A
to C). Thus, SOSTDC1-expressing T cells are a
distinct subpopulation of TFHcells that develop
from SOSTDC1–TFHcells and lose their ability
to provide help for B cells.
During the course of keyhole limpet hemo-
cyanin (KLH) immunization,Sostdc1deficiency
substantially increased the percentage of GC
B cells and serum levels of KLH-specific im-
munoglobulin G2c (IgG2c) and IgG2b while
leaving the TFHcell population unchanged
(fig. S6, A to C). By contrast, TFRcells were
significantly reduced as a percentage of total
cells and in comparison with TFHcells (fig. S6,
C and D). Furthermore, we adapted a cotrans-
fer mouse model and foundSostdc1deficiency
in donor OT-II cells blocked TFRcell generation
and consequentially enhanced GC responses
(Fig. 3, A to C).Sostdc1deficiency reduced the
percentage, but not number, of TFHcells (Fig.
3B and fig. S7, A and B). In the context of in-
fluenza viral infection,Sostdc1–/–mice displayed
substantially reduced weight loss, viral loads,
and lung tissue damage compared with wild-
type (WT) mice (Fig. 3, D to F). Consistent with
these KLH immunization data,Sostdc1defi-
ciency largely increased GC responses, con-
comitant with reduced TFRcell numbers and
impaired expression of ICOS and CTLA4 by
TFRcells (Fig. 3, G and H, and fig. S8, A to C).
Thus,Sostdc1ablation results in TFRdefects,
which, in turn, enhances humoral immunity
against viruses.
To delineate the roles of TFHcell–and FRC-
derived SOSTDC1 in TFRcell differentia-
tion, we first generated mixed bone marrow
chimeras by transferring WT bone marrow
cells toSostdc1–/–recipient mice and vice versa.
This demonstrated that SOSTDC1-expressing
cells of both hematopoietic and nonhemato-
poietic origin support TFRcell generation. (fig.
S9, A to D). Furthermore, bone marrow chimeras
reconstituted with either WT +Sostdc1–/–or
Bcl6fl/fl/Cd4Cre+Sostdc1–/–bone marrow cells
revealed that TFH-derived SOSTDC1 was crit-
ical for TFRcell formation (fig. S10, A to C).
Thus, SOSTDC1-expressing TFHcells may func-
tion with nonhematopoietic FRCs to support
TFRcell generation through SOSTDC1.
To rule out the possibility that defective
features of TFRcells bySostdc1ablation are
potentially inherited from thymic T cells, weanalyzed and compared T cell populations
and activation status between WT andSostdc1–/–
mice. The percentages of CD4+, CD4+CD8+,
and CD8+T cells in the thymus and Foxp3+
Tregcell in the lymph nodes were unaffected
bySostdc1ablation (fig. S11, A and B). Ad-
ditionally, in chimeric mice reconstituted
with WT andSostdc1–/–bone marrow,Sostdc1
deficiency did not affect thymic T cell devel-
opment and peripheral T cell homeostasis
(fig. S11, C and D).
SOSTDC1 inhibits the canonical WNT–b-
catenin pathway ( 17 – 19 ). The most pronounced
difference between SOSTDC1–and SOSTDC1+
TFHcell subsets was the overexpression of
WNT ligand genes in SOSTDC1–TFHcells,
whereas WNT-antagonistic genes were elevated
in SOSTDC1+TFHcells (Fig. 4A). Given that
canonical WNT signaling inhibits Tregcells
( 20 , 21 ), we hypothesized that the defective
TFRcells observed inSostdc1deficiency are due
to the dysregulation of the WNT–b-catenin
signaling axis.Sostdc1ablation increasedb-catenin
levels in TFRcells significantly more than in
TFHand Foxp3+CXCR5–Tregcells (Fig. 4B and
fig. S12A). Consistent with this, the global
transcriptional changes bySostdc1ablation inWuet al.,Science 369 , 984–988 (2020) 21 August 2020 3of5
ABCFD EG
16.1 ± 1.222.1 ± 1.8TFR cell
WTTFH cell
WT11.8 ± 2.1GC B cell
WTPD-1CXCR59.4 ± 0.500102
102103103104104105105GL7FAS43.3 ± 7.300102102103103104104105PD-1^105CXCR53.78 ± 0.500102
1021031031041041051050
0102
102103103104104105105
CD4Foxp3Donor CD4+ T cell6.6 ± 0.291.1 ± 1.0WT5.35 ± 0.300102
1021031031041041051050.846 ± 0.0710
ICOS0 2 103 104 105610
216CTLA40102 103 104 1051818
767WTGated on
CD4+ CD44hi Foxp3+HSostdc1–/–00102
102103103104104105105
CD4Foxp3Donor CD4+ T cell6.6 ± 0.291.1 ± 1.0Sostdc1–/–05101520(^25)
0
2
4
6
(^8)
0
5
10
15
NS
0.0
0.5
1.0
1.5
2.0
- 0
10
20
(^30)
0
10
20
30
40
(^50) NS
0
20
40
60
0
5
10
15
20
25
TFR
(% of Foxp3
+)
Treg
(% of CD4
+)
Treg
cells (1 × 10
3 )
TFR
cells (1 × 10
3 )
WT
Sostdc1–/–
TFH
(% of CD4
+)
TFH
cells (1 × 10
3 )
WT
Sostdc1–/–
Sostdc1–/– GC B cells (%)
GC B cells (1 × 10
3 )
WT
Sostdc1–/–
Body weight %WTSostdc1–/–
0
100
200
300
400
HA
mRNA level
1 2 3 4 5 6 7 8 9 10 11 12Days
Sostdc1–/–
60
70
80
90
100
110
120
WT
Sostdc1–/–
Sostdc1
–/–
PD-1
CXCR5
0
2
4
(^6) *
12
14
16
18
20 NS
0
1
2
3
0
20
40
60
80 NS
0
2
4
6
8
0
5
10
15
(^20)
TFR
cells (%)
Treg
(% of CD4
+)
Treg
cells (1 × 10
4 )
TFR
cells (1 × 10
4 )
ICOS (MFI)(1 × 10
2 )
CTLA4 (MFI)
(1 × 10
2 )
Naive T
Sostdc1–/–
TFR WT
WT
Sostdc1–/–
WT
Sostdc1–/–
80
TFR
Fig. 3. TFHcell–derived SOSTDC1 is required for TFRcell generation.
(AtoC) Sorted Foxp3-GFP+nTregand B1.8 B cells were cotransferred with
naïve WT orSostdc1–/–OT-II cells intoRag1–/–recipient mice, followed by
immunization with NP-OVA and CFA subcutaneously for 7 days. Flow
cytometric analysis and quantification of TFRand Tregcells (A), TFHcells (B),
andGCBcells(C)indLNs.(DtoH)WTandSostdc1–/–mice were
infected intranasally with influenza virus A/PR8 for 15 days. Their body
weights (D) were measured and lung viral titers were assessed by
hemagglutinin (HA) gene expression with qRT-PCR (E). (F) Representative
hematoxylin and eosin images of lung sections. Scale bars, 200mm.
Flow cytometric analysis and quantification of TFRcells numbers (G) as
well as TFRcell ICOS and CTLA4 expression (H) in lung dLNs. Red, WT TFR
cells; blue,Sostdc1–/–TFRcells; gray, naïve CD4+T cells. Data are
representative of at least two independent experiments, with three to five
mice per group. Error bars indicate SEM. Statistical tests: two-way ANOVA
[(A) to (D)] and Student’sttest [(E), (G), and (H)]; P<0.05,**P<0.01.
RESEARCH | REPORT