ofSostdc1EGFP/Bcl6RFPdual-reporter mice dem-
onstrated that SOSTDC1+TFHcells (~5%)
coexisted distinctly from SOSTDC1–TFHcells
(~10%) in the draining lymph nodes (dLNs)
(Fig.1D).Furthermore,mostSOSTDC1+TFH
cells were able to maintain their phenotype
during a recall response (Fig. 1E). Thus, a dis-
tinctive subpopulation of TFHcells exists that
is defined by SOSTDC1 expression.
Gene expression analysis revealed that the
SOSTDC1+TFHcells were distinct from those
of SOSTDC1–TFHand non-TFHcells (Fig. 2,
A and B). In comparison with non-TFHcells,
although both SOSTDC1+and SOSTDC1–TFH
cells expressed equivalent levels of the TFHcell
signature genesAscl2,Bcl6, andCxcr5and
reduced expression ofGata3,Ifng,Rorc,and
Tbx21,SOSTDC1+TFHcells distinguished them-
selves from SOSTDC1–TFHcells by overexpres-
sion ofSostdc1andZfp703and reduction ofIl4,
Il21,andCd154, as well as WNT-related genes,
such asWnt3aandLrp5(Fig.2C).Single-cell
RNA sequencing (scRNA-seq) analysis con-
firmed differential transcriptome between
Sostdc1+(cluster 4) andSostdc1–(cluster 2)
TFHcells (fig. S4, A and B). Further pseudo-temporal ordering analysis demonstrated a
developmental transition fromSostdc1–to
Sostdc1+TFHcell (fig. S4C). In vivo fate map-
ping showed thatSostdc1+TFHcell appears to
develop from plastic precursor SOSTDC1–TFH
cells (fig. S4D). Although both populations
of SOSTDC1+and SOSTDC1–TFHcells were
predominantly distributed in follicle and T–B
cell interface regions, higher percentages of
SOSTDC1+TFHcells were located in follicles
and GC regions than SOSTDC1–TFHcells (fig.
S4E). Functionally, unlike SOSTDC1–TFHcells,
SOSTDC1+TFHcells were unable to help B cellsWuet al.,Science 369 , 984–988 (2020) 21 August 2020 2of5
(^1) Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, P. R. China. (^2) State Key Laboratory of
Trauma, Burns and Combined Injury, Third Military Medical University (Army Medical University), Chongqing 400038, P. R. China.^3 Institute for Systems Biology, Seattle, WA 98103, USA.
(^4) Institute for Immunology and School of Medicine, Tsinghua University, Beijing 100084, P. R. China. (^5) Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan,
250012, P. R. China.^6 Medical Research Institute, School of Medicine, Wuhan University, Wuhan, 430071, P. R. China.
*Present address: National Research Center for Translational Medicine (Shanghai), Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China.
†Corresponding author. Email: [email protected] (X.L.); [email protected] (X.-W.B.); [email protected] (Y.W.)
A CD4+CD44hi T cells
105
104
103
102
0
0
0
0
102
102
102
103
103
103
104
104
104
105
105
105
T Zone
GC
Follicle
GC
SOSTDC1-EGFP
BCL6-RFP
TTTTTTTTTTTTTZonZonZonZonZ eeeeeeeeeeeeeeee
GCGCGCGCGCGCGC
FllFollFollFollFollFollFollFollFollFolloicleicleililicleicleicleicleicle
GCGCGCGCGCGGCGCCCCCCCCC
2
1
0
-1
-2
Non-T
FH
SOSTDC1
- SOSTDC1
Non-TFHSOSTDC1-EGFP+ TFHSOSTDC1-EGFP–TFHB50.97 % of the variance explainedNon-TFH- TFH Non-TFH (2)
- T
FH (2)
- T
SOSTDC1+ TFH (2)SOSTDC1+ TFHPCA using 10,870 genes37.12 % of the variance explainedControl0102 103 104 105204060801000CDE ControlNon-TFHWnt1 (109)
Wnt3a (166)
Bmp6 (419)
Wnt4 (522)
Bmp1Fzd9 (627) (538)
Wnt10a (694)
Bmp8a (978)
Wnt9a (1293)
Fzd1 (1369)
Wnt8bFzd8 (1554) (1534)
Wnt10b (1568)
Bmp4 (1609)
Fzd10 (1656)
Wnt3 (1940)
Bcl6 (2761)
Cxcr5 (2777)
Cdc20 (3490)
Cdkn1b (3664)
Cdc25cCdk12 (3953) (3745)
Cenpb (4022)
Top2a (4822)
E2f2 (5078)
Sostdc1 (5311)
Ifngr1Ccl5 (6113) (5978)
Il1rn (6246)
Il9 (6252)
Il11 (6355)
Ccl17 (6356)
Il21r (6427)
Cxcl1 Cxcl10(6429) (6829)
Il17re (6891)
Il23r (7154)
Foxp3 (7597)
Il2ra (7632)
Il4ra (7763)
Ccr2 (8104)
Cxcr6 (8125)
Cdk1Mki67 (9784) (9951)
Birc5 (10437)500-50-100 -50 0 50 10005101520NSNS **NS
********051015******0510152025
******Relative mRNA level Relative mRNA levelRelative mRNA levelTbx21 Gata3 Rorc Wnt3a Lrp5 Zfp703Cxcr5 Bcl6 Sostdc1 Il4 Il21 Ifng Icos Cd154EGFP+ TFHSOSTDC1-
EGFP–TFH
0.065 15.9 1.37GL7IgG10510152025 **
0510152025
**0204060(^80) **
IgG1
- cells (%)
IgG1
MFI (1 × 10
2 )
GL7 MFI (1 × 10
2 )
Control
SOSTDC1-EGFP+ TFH
SOSTDC1-EGFP–TFH
CTV
% of Max
0
20
40
60
80
(^100)
Proliferated cells (%)
Control
SOSTDC1
SOSTDC1
SOSTDC1-
SOSTDC1-EGFP+ TFH
SOSTDC1-EGFP–TFH
SOSTDC1-EGFP+ TFH
SOSTDC1-EGFP–TFH
SOSTDC1-EGFP+ TFH
SOSTDC1-EGFP–TFH
Fig. 2. SOSTDC1-expressing TFHcells represent a distinct subpopulation
of TFHcells that do not help B cells to promote antibody production.
(AtoC) RNA-seq assessment of sorted CD44hiCD4+BCL6-RFP+SOSTDC1-
EGFP–TFHcells, BCL6-RFP+SOSTDC1-EGFP+TFHcells, and BCL6-RFP–
SOSTDC1-EGFP–non-TFHcells fromSostdc1EGFP/Bcl6RFPdual-reporter mice
that were immunized subcutaneously with KLH and CFA for 7 days.
(A) Heatmap of genes expressed differentially in these three populations of
cells. (B) Principle components analysis (PCA) of their transcriptomes.
(C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
measurement of gene expression by these three populations. NS, no
significance. (DandE) Assessment of the role of TFHcells in regulating B cell
responses. Non-TFH(control), SOSTDC1-EGFP–TFH, and SOSTDC1-EGFP+
TFHcells were sorted fromSostdc1EGFPmice that received immunization
with KLH and CFA subcutaneously for 7 days and cocultured separately
with B220+IgD+B cells for 6 days. (D) Flow cytometric analysis of class-
switched (IgG1+GL7+) B cells from cocultures. MFI, mean fluorescence
intensity. (E) B cell proliferation measured by Cell Trace Violet (CTV) at
day 6. Max, maximum. Data represent at least two independent experiments
with five to seven mice per group. Error bars indicate SEM. Statistical tests:
one-way analysis of variance (ANOVA). P<0.01.
RESEARCH | REPORT