the same experiment longitudinally using
an iLN imaging window. Corroborating our
previous data, Foxp3 expression began to
rise prior to the onset of GC collapse, peak-
ing at about 10% of transferred cells at day 18
postimmunization (Fig. 5, E to G). Thus, up-
regulation of Foxp3 by TFHcells contributes
to the surge in Foxp3+cells that takes place in
end-stage GCs.
Late-GC Foxp3+T cells display an intermediate
phenotype between TFHand TFRcells
To determine what effect Foxp3 expression has
on TFHcells, we carried out whole-transcriptome
single-cell RNA sequencing (scRNA-seq) on
T cells sorted from individual photoactivated
GCs at day 10 or 20 postimmunization as de-
scribed earlier (fig. S1). For reference, we also
sorted a sample of Foxp3+TFH-phenotype T cells
converted from adoptively transferred naïve
precursors (Fig. 5) and one plate of Foxp3+
T cells photoactivated in the T-zone (table S1).
The 968 cells that passed the quality threshold
fell into six major clusters (Fig. 6A and fig. S6,
AtoD).RFP+T cells were distributed across
clusters 1 and 2 (TFH1 and TFH2, respectively),
3 (Treg/resting), and 4 (activated Treg), indica-
tive of heterogeneity among this population
(Fig. 4B). As expected, T-zone Tregswere found
almost exclusively in clusters 3 and 4. By con-
trast, RFP+cells from photoactivated GCs were
also often found in the two TFHclusters, as were
naïve transfer-derived CXCR5+PD-1hiFoxp3+
cells (Fig. 6B). This was confirmed when only
cells with detectableFoxp3mRNA were analy-
zed (fig. S6E). Thus, two major populations of
Foxp3+T cells are present in GCs, those that
are TFH-like (clusters 1 and 2) and those that
resemble T-zone Tregs(clusters 3 and 4). The
relative absence of prototypical Tregtranscript
Il2raexpression (encoding for CD25) (fig. S6C)
in Foxp3+T cells from clusters 1 and 2 suggested
thatthesecellsmayresembleapreviouslyde-
scribed GC-resident CD25–Foxp3+population
(“GC-TFR”) with a hybrid TFH/Tregphenotype
( 31 ). Indeed, transcriptional signature analysis
showed that Foxp3+cells within TFHclusters
1and2resembleGC-TFRcells, whereas cluster
4 Foxp3+cells resemble the canonical CD25+
TFRpopulation (Fig. 6C). Thus, although clus-ter 1 and 2 Foxp3+T cells are TFH-like, expres-
sion of Foxp3 shifts these cells toward a
transcriptional state similar to that of CD25–
GC-TFRcells.
To determine whether this hybrid transcrip-
tional state is also observable in Foxp3+cells
known to be derived from Foxp3–TFHprecur-
sors, we took two approaches. We first com-
pared gene expression between Foxp3–TFH
cells in photoactivated late GCs and spiked in
Foxp3+TFHcells derived from transferred naïve
T cells. This comparison showed evidence of
acquisition by naïve-transferred Foxp3+cells
of both the CD25–GC-TFRprogram ( 31 ) and of
other Treg-associated signatures ( 32 ) (Fig. 6D),
as well as loss of expression of T cell help-
associated genes such asIl21( 33 ) andCd40lg
( 34 ). As a second, more strict approach, we
defined Foxp3+T cells of TFHorigin by deter-
mining the TCR sequences of day 20 GC T cells
using scRNA-seq data confirmed by long-read
polymerase chain reaction (PCR)–based se-
quencing. Eleven clonal expansions contained
both Foxp3+and Foxp3–cells. With few ex-
ceptions, Foxp3+and Foxp3–cells within theseJacobsenet al.,Science 373 , eabe5146 (2021) 16 July 2021 4 of 13
B T:B contacts, T:B borderE T:B contacts, GCF Foxp3+ T cell velocity G Foxp3+ T:OT-II contactsA Day 2, T:B border FoxP3 OT-II (+Ca2+) B1-8hi
1:06 4:26 7:46 10:331:06 6:04 8:51 12:42 17:080102030Duration of interactionwith B cell (min)OT-II Foxp3+P < 0.00010481216Duration of interactionwith B cell (min)10 14/151 0 14/15P = 0.0004Contacting cell Days post-boost
OT-II Foxp3+C Day 10, germinal center FoxP3OT-IIB1-8hi
0:33 2:14 4:27 5:3420:38 21:11 22:51 24:32D Day 14/15, germinal center FoxP3OT-IIB1-8hi8:20 12:48 16:42 20:02 22:4928:57 30:37 32:50 36:11 38:24Days post-boostDuration of interaction with OT-II T cell (min)
0481216 P = 0.09710 14/15
Days post-boost10Mean track
velocty (μm/min)P = 0.0006051015202514/15Fig. 3. Dynamics of the interaction between B cells and Foxp3+T cells.(A) CFP
+B1-8hiB cells and dsRed+GCaMP3+OT-II T cells were adoptively transferred into
Foxp3GFPhosts, which were then immunized in the footpad with 8mg of NP-OVA
in alum. Intravital imaging on pLNs was performed 48 hours later. The time series
show interactions (indicated by arrowheads) between Foxp3+T cells (green) and
B1-8hiB cells (blue). OT-II T cells are in red (turning orange or yellow when fluxing
calcium). Tracks of interacting Foxp3+cells are shown. (B) Quantification of
interactions between B1-8hiB cells and OT-II or Foxp3+T cells at different time
points. Each symbol represents one interaction. Only interactions lasting two frames
or longer were included. A dotted line is placed at 4 min for reference. (Cand
D) Intravital imaging on pLN was performed on days 10 or 14 and 15 postboost.
The experimental setup is as in Fig. 1A. The time series is as described in (A).
(E) Quantification of interactions in (C) and (D), as described in (B). (F) Mean track
velocity for Foxp3+T cells at early and late time points. Each symbol represents
one track. (G) Quantification of interactions between host Foxp3+T cells and
transferred OT-II T cells at different time points. Details are as in (B). Scale bars:
10 mm. Three movies from three independent experiments were analyzed for each
dataset. (B) and (E) to (G) show pooled data, with each dot representing a cell and
a bar representing the mean. Time stamps are in reference to the start of the
movie (see Movies 1, 2, and 3).Pvalues are for StudentÕsttest.RESEARCH | RESEARCH ARTICLE