“mixed”clones remained predominantly with-
in TFHclusters 1 and 2 and were not found
within cluster 4, likely to include most of the
canonical TFRcell population (Fig. 6E). How-
ever, despite the small number of cells in this
analysis, Foxp3+cells showed statistically de-
tectable modulation of GC-TFHsignature genes
and of a set of 30 genes down-regulated by
Foxp3+TFHderived from transferred naïve
precursors (Fig. 6F and fig. S7). This included
detectable down-regulation ofIl21, whereas
Cd40lgmRNA was not well captured in this
sample (Fig. 6F). Thus, althoughFoxp3expres-
sion is insufficient to fully convert late TFHcells
into the Tregor TFRphenotype, it is associated
with the induction of Treg-associated transcrip-
tional changes, suggesting that Foxp3 may play
a functional role in these cells.
Foxp3 up-regulation by TFHcells promotes
contraction of late GCs
To determine whether acquisition of Foxp3 by
late-stage TFHcells can promote GC shutdown, we
generated mice carrying an inducibleRosa26Foxp3
allele, where near-physiological expression of
Foxp3 protein, followed by a GFP reporter, is
conditional upon removal of a loxP-flanked stop
cassette by cre-mediated recombination (Fig. 7A
and fig. S8). To acutely induce Foxp3 expression
by TFHcells, we adoptively transferred CD4+
T cells fromRosa26Foxp3CD4-CreERT2 OT-II
mice into allelically marked CD45.1 P25 TCR-
transgenic hosts, which we then immunized in
the footpad with NP-OVA in alhydrogel (Fig.
7A). This protocol generates larger, longer-lived
GCs, in which roughly 70% of TFHcells derive
from the donor mouse. Notably, OT-II TFHcells
are refractory to spontaneously up-regulating
Foxp3expression even at later time points (fig.
S9), in line with previous studies of TFRdiffer-
entiation using this TCR ( 13 , 35 ). Tamoxifen
administration led to detectable expression
of Foxp3 protein in about 40% of transferred
T cells (corresponding to about 30% of all TFH
cells), at levels that matched those of T cells
naturally expressing Foxp3 in the TFHgate (Fig.7B).Rosa26Foxp3-expressing cells up-regulated
Tregmarkers cytotoxic T lymphocyte–associated
protein 4 (CTLA-4) and, to a lesser extent,
glucocorticoid-induced tumor necrosis factor
receptor–related protein (GITR), but not CD25
(Fig. 7B and fig. S10). scRNA-seq of OT-II TFH
cells forced to express Foxp3 compared with
control OT-II CD4-CreERT2+T cells lacking the
Rosa26Foxp3allele recapitulated the changes
observed during physiological up-regulation of
Foxp3+within mixed Foxp3+-Foxp3–TFHclones
(Fig. 6, E and F). Although expression of Foxp3
alone was again not sufficient to segregate TFH
cells into different nearest-neighbor clusters
(fig. S10C), Foxp3+cells showed clear changes in
the expression of both“naïve transfer”(fig. S7)
and“GC-TFH”( 31 ) signatures, including limited
but detectable loss ofIl21andCd40lgmRNAs
(Fig. 7C and fig. S10D). Thus, forced expression
of Foxp3 induces TFHcells to modulate ex-
pression of a Treg-associated transcriptional
program similar to that acquired under phys-
iological conditions. Most importantly, ectopicJacobsenet al.,Science 373 , eabe5146 (2021) 16 July 2021 5 of 13
CDEEarlyLate020406080% of expanded clones includingboth Foxp3+ and Foxp3- cells
P = 0.048 P = 0.0200510152025P = 0.004Expanded Foxp3+-only clones(% of all sequenced cells)EarlyLate020406080100P = 0.006% Foxp3+
cells(of all sequenced cells)EarlyLateAIndex-sort single
photoactivated T cellsFoxp3-RFPPAGFP (activated)105
104
103
0
0 103104105Sorted cellsFoxp3– Foxp3+ Expanded clones, homogeneous Expanded clones containing both Foxp3+ and Foxp3– cellsB111/116 127/151 67/94 55/7872/134 35/55 44/61 73/13693/114
Days 9-11Sequence Days 18-20
TCRα/β
Foxp3–
Foxp3+Foxp3+ Tfh
overlapTCRoverlapNo TCR102103104105102103104105CXCR5PD-1100 8859 74102103104105102103104105102103104105102103104105(^0102103104105102103104105)
10
20
30
% of Foxp3
- cells in clones
that include Foxp3
- cells
EarlyLateTCR
overlapNo TCR
overlap080
60
40
20100Foxp3+
Tfh% CXCR5+PD-1hiP = 0.017137/163Fig. 4. A subset of late-GC Foxp3+T cells arises through up-regulation
of Foxp3 by TFHcells.(A) Experimental setup. RFP+and RFP–T cells from
single photoactivated GCs obtained from Foxp3RFP+PAGFP-tg mice at days 9
to 11 (early) or 18 to 20 (late) after primary immunization with NP-OVA
were index-sorted for TCR-aand -bsequencing. (B) Clonal distribution of T cells
within single GCs. Each pie chart represents one GC. Numbers are (number
of clones)/(number of cells sequenced). Expanded clones (defined as those
found more than once within the same GC) are colored in gray when including
only one cell type or blue when including both cell types, with the cell type
composition of the clone indicated in the outer circle. Additional pie charts
can be found in fig. S3; full TCR sequences are available as data S1.
(C) Quantification of data in (B), comparing early and late time points. Each
symbol represents one GC. Includes additional late GCs not shown in (A).
(D) Expression of TFHmarkers among RFP+and RFP–cells stratified by
TCR overlap, obtained from index-sorting data.“Overlapping”cells are
defined as those belonging to clones that contain both RFP+and RFP–cells.
(E) Quantification of data in (D). Each symbol represents one GC. Data
are for six GCs from five independent experiments (days 9 to 11) and
10 GCs from nine independent experiments (days 18 to 20). Bar indicates
the median.Pvalues are for Student’sttest.RESEARCH | RESEARCH ARTICLE