Science - USA (2020-08-21)

TFR,butnotCXCR5–Treg, cells were positively
correlated with that inb-catenin–stabilized
Tregcells ( 21 ) (Fig. 4C and fig. S12, B to D).
Specifically,Sostdc1deficiency significantly
up-regulated the expression of effector cyto-
kines (Il4,Il5,Il6,Il13,Il21, andIfng) and
down-regulated transcriptional repressors
(Nr4a1,Id3,andFoxp4)inTFRcells (Fig. 4D).
Furthermore,Sostdc1ablation enrichedb-catenin–
lymphoid enhancer factor–Tcellfactorbind-
ing motif while reducing KLF family binding
in TFRcells (fig. S12E). Last, there were com-
parable levels of bone morphogenetic protein
(BMP) pathway–relatedSmad4andphos-
phorylated Smad1 and Smad5 in WT and
Sostdc1–/–TFRcells (fig. S12F), excluding the
possible correlation of SOSTDC1 with the BMP
signaling cascade ( 22 , 23 ). Thus,Sostdc1ab-
lation predominantly augments theb-catenin–
controlled transcriptome, which blocks TFRcell
differentiation.

To specify the role ofb-catenin in TFRcells,

we generated chimeric mice containing

WT orCtnnb1ex3/Ert2CrenTregcells in which

b-catenin can be stabilized by tamoxifen (fig.

S13A). Assessment of donor cells revealed

that stabilization ofb-catenin impaired TFR

cell generation significantly (fig. S13B). Addi-

tionally, in chimeras transduced with WT

andCtnnb1fl/fl/Cd4Crebone marrow,Ctnnb1fl/fl/

Cd4Crebone marrow–derived TFRcells pre-

dominated over WT bone marrow–derived

counterparts(fig.S13C).Thus,b-catenin

plays a negative role in regulating TFRcell

differentiation.

SOSTDC1-EGFP+cells were localized prox-

imally to Foxp3+T cells in B cell follicle and

T–B cell border regions. Furthermore, more

than 60% of Foxp3+T cells were surrounded

by at least one SOSTDC1-EGFP+neighbor cell

with a distance of less than one cell diameter

(Fig. 4E), suggesting that SOSTDC1+cells func-

tion by aiding nearby TFRcells in B cell follicle

and T–B cell border regions. Last, in vitro

coculture assays demonstrated that SOSTDC1+

TFHcells helped maintain the TFRcell pheno-

type (fig. S14). Thus, SOSTDC1-expressing TFH

cells have the capacity to promote TFRcell

formation and maintenance.

Previous studies have shown that SOSTDC1

is capable of modulating the WNT–b-catenin

signaling pathway through WNT receptors,

including LRP4, LRP5, and LRP6 ( 17 , 19 ). Only

Lrp5 and Lrp6 were highly expressed in TFR

and Tregcells (fig. S15A). When stimulating

T cells with Wnt3a plus increased dosage of

SOSTDC1, SOSTDC1 attenuated the levels of

b-cateninandphosphorylatedLRP6inadose-

dependent manner (fig. S15B). Furthermore,

double disruption of LRP5 and LRP6 appreciably

rescued both Foxp3+Tregcell and Foxp3+CXCR5+

TFRcell subsets in the context of heightened

WNT ligands (fig. S15, C to E). Thus, SOSTDC1

cangovernTFRcell differentiation by neutral-

izing extrinsic WNT ligands.

Uncontrolled WNT–b-catenin signaling plays

a critical role in autoimmune diseases ( 20 , 24 – 27 ).

SOSTDC1, produced by a subpopulation of

TFHcells and T–B cell interface–resident

stromal FRCs, is able to promote commit-

ment to the TFRcell fate by means of the

blockade of WNT stimuli. Both this TFHcell

subpopulation and resident FRCs form a

negative regulatory layer to curtail humoral

immunity (Fig. 4F). Consequently, manipula-

tion of the reciprocal SOSTDC1–WNT signal-

ing axis may have clinical benefits for vaccine

development and the treatment of autoim-

mune diseases.

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Wuet al.,Science 369 , 984–988 (2020) 21 August 2020 4of5

B

TFR

• T
  FH

SOSTDC1+ TFH

Wnt signaling Antagonistic Wnt

Enrichment plot: WNT SIGNALINGPATHWAY_GOBP_GO:0016055

0102 103 104 105

1277

1798

0102 103 104 105

613

Non-T 877

FR

Treg

A Isotype IgG

Sostdc1–/–

WT

10

15

(^20) *
6
8
10
12

of T
FR
(1 ×10
2 )
of T
reg
(1 × 10
2 )
WT
Sostdc1–/– 0.70.60.5
0.40.3
0.20.1
-0.10.0
Enrichment Score (ES)
P = 0.0001
FDR = 0.0001
Up (Sostdc1–/–vs. WT) Down
P = 0.0001
FDR = 0.0001
Up (Sostdc1–/–vs. WT) Down
0.0
-0.1-0.2
-0.3-0.4
-0.5-0.6
-0.7
Enrichment Score (ES)
TFR
Ctnnb1
CA
vs
.WT
Geneset Up
Geneset Down
C
-7-6-5-4-3-2-1 01234567
-7
-6
-5
-4
-1
0
1
2
3
4
5
6
7
Log
(Ctnnb1 2
CA
/ WT)
Log 2 (Sostdc1–/–/ WT)
Ccl1
Ccl4 Ccl3
Il10
Il13 Il21
Il5 Il4
Il6
Cd40lg
Cxcr6
Dusp7
Ezh2
Foxp4
Gzmk
Id3
Ifng
Irf7
Jun
Lif
Nr4a1
Socs1
Tnfsf14
Tnfsf8
-2
-3
D
T Zone
Follicle
Interfollicle
0
20
40
60
80



Foxp3

• cells (%)
  0:1 1:1-2 1:3-5
  SOSTDC1-EGFP+:Foxp3+
  IgD /
  SOSTDC1-EGFP
  Foxp3
  TFR
  TFR
  Germinal
  center
  B follicle
  Inter
  follicle
  B
  Sos–
  Sos–
  Sos+
  Sos+ TFHSOSTDC1
  FRCs
  nTreg
  T zone
  Non-TFH
  T
  T
  DC
  WNT
  E F
  SOSTDC1
  TFH
  Fig. 4. SOSTDC1 promotes TFRcell generation through blockade of the WNT–b-catenin pathway.
  (A) Gene set enrichment analysis (GSEA) of transcriptomes in SOSTDC1-EGFP–and SOSTDC1-EGFP+TFH
  cells. (B)b-catenin expression in TFRand non-TFRTregcells from WT andSostdc1–/–mice immunized
  subcutaneously with KLH and CFA for 7 days. (CandD) RNA-seq analyses of GITRhiTFRcells and
  GITRhinon-TFRTregcells were sorted from WT orSostdc1–/–mice immunized subcutaneously with KLH
  and CFA for 7 days. These populations were valuated with Foxp3 staining. (C) GSEA of altered genes
  in TFR(Sostdc1–/–versus WT) in the context of gene set from Treg(Ctnnb1CAversus WT) ( 21 ). FDR, false
  discovery rate. (D) Gene expression scatter plot of 8245 genes altered similarly between TFR(Sostdc1–/–
  versus WT) and Treg(Ctnnb1CAversus WT) cells. Red, up-regulated effectors; green, down-regulated
  transcriptional repressors. (E) Immunofluorescent staining and assessment of Foxp3+and EGFP+cells
  in dLNs sections fromSostdc1EGFPmice immunized with KLH and CFA subcutaneously for 7 days.
  Red, Foxp3; blue, IgD; and green, SOSTDC1-EGFP. Scale bars, 200mm. (F) Schematic model for the
  differentiation and function of SOSTDC1–TFHcells. The 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, *P<0.05,**P<0.01.
  RESEARCH | REPORT