Science - USA (2021-07-16)

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expression of Foxp3 in peak GC TFHcells led
to a roughly 60% reduction in GC size com-
pared with that in control mice that did not
receive tamoxifen or tamoxifen-treated mice
receiving Cre+control T cells (Fig. 7C). Thus,
Foxp3expression by TFHcells is sufficient to
both promote a Treg-like phenotype in TFHcells
and to accelerate the contraction of the GC
reaction.


Discussion


Our data support a model in which the con-
traction and eventual shutdown of late-stage
GCs is driven at least in part by a surge in
Foxp3+GC T cells. This surge is partly due to
acquisition of Foxp3 by TFHcells, which join
preexisting tTreg-derived TFRand GC-TFRpop-
ulations to dramatically increase Foxp3+T cell
density in late GCs.
Two major considerations underlie our con-
clusion that late-GC Foxp3+T cells originate
partly from TFHprecursors. First, intravital


imaging showed that the quality of inter-
actions between Foxp3+T cells and GC B cells
changes markedly with time, from an early
preponderance of the brief interactions typical
of TFRcells ( 23 ) to the appearance of the long-
lived entanglements typical of TFHcells ( 26 )
at the time of the Foxp3 surge. This suggests
either that TFRcells undergo a dramatic change
in dynamic behavior at late stages or, more
likely, that they are joined in their ranks by a
second population of Foxp3+T cells with
TFH-like behavior. Second, and most impor-
tant, end-stage Foxp3+T cells share TCR se-
quences with Foxp3–TFHcells, indicating a
common precursor. Our finding that adoptively
transferred Foxp3–naïve T cells up-regulate
Foxp3 in late but not early GCs suggests that,
in this particular case, the direction of change
is from Foxp3–to Foxp3+, rather than in the
opposite direction as observed in early GCs
using a fate-mapping model ( 36 ). This timing
also suggests that late-GC Foxp3+TFHcells are

unlikely to be derived from pTregcells, because
these would be expected to be present in
earlier-stage GCs as well ( 30 ).
Critically, expression ofFoxp3is not suffi-
cient to trigger TFHcells to adopt a full-fledged
TFRor Tregphenotype. Instead, TFHcells
resemble more closely a previously described
CD25–“GC-TFR”population ( 31 ), which has an
intermediate phenotype between TFHand
canonical TFRcells. Given that GC-TFRcells
are present also in early GCs and are thought
to derive from TFRprecursors ( 31 ), it is likely
that late-GC Foxp3+TFHcells are not identical
to GC-TFRcells, but rather assume some of
their transcriptional characteristics and pos-
sibly regulatory properties.
It is unclear what triggers the up-regulation
of Foxp3 by TFHcells in end-stage GCs. TGF-b
was capable of inducing Foxp3 expression in
TFHcells cultured in vitro (fig. S5) and has been
shown to be active within the GC environment
in vivo ( 37 ), suggesting that this cytokine at

Jacobsenet al.,Science 373 , eabe5146 (2021) 16 July 2021 6 of 13


Fig. 5. Up-regulation of Foxp3
in late GCs by adoptively
transferred naïve T cells.
(A) Experimental setup.
(B) Sorted RFP+GFP(Foxp3)–
CD4+T cells were transferred
into P25 TCR-tg recipients, which
were immunized with NP-OVA
in alum and analyzed by
multiphoton microscopy at
days 10 (left) or 19 and
20 (right) postimmunization,
as in fig. S3D. Images show
single optical slices of GCs.
GC cross section (dashed white
line) was defined based on
in vivo FDC (CD35) labeling.
RFP+GFP+cells are indicated
as yellow dashed circles
and magnified in the insets.
Rendering shows full GC
volumes, with RFP+cells shown
as smaller red spheres and
RFP+GFP+cells shown as larger
green spheres. Scale bars:
40 mm. (C) Quantification of
data in (B). (D) Representative
flow cytometry plot from a
late GC generated as in (A).
(EandF) Longitudinal imaging
of transferred RFP+GFP(Foxp3)–
T cells. Details are as in (A).
iLN window was mounted on
day 8 postimmunization. Data
are for a single experiment.
White dashed lines and yellow
dashed circles are as in (B).
Scale bars: 50mm. (G) Quantifi-
cation of data in (E) and (F).Pvalues are for Student’sttest. In (C), each dot represents one GC from three mice in three independent experiments (early) and
five mice in three independent experiments (late). Bar indicates the median. Data in (E) and (F) are for a single experiment.


ii.

ii.

i.

Foxp3

cells in GC+

(% of transferred T cells)

GC volume (% of max)

Days post-immunization

10 12 14 16 18 20

0

20

40

60

80

100

0

5

10

15

Transfer Foxp3 CD35

Day 10

i. ii. i.

i.

ii.
i.

i.

i.

Day 13 Day 14 Day 18

F Day 10 Day 13 Day 14 Day 16 Day 18 Day 21

Transfer Foxp3 CD35

B Naïve T cell transfer/cross-sectional imaging

% GFP

+ of transferred cells
0

5

10

15

20

P < 0.0001

Early Late

C

E Naïve T cell transfer/longitudinal imaging G

i.

Day 19

D

12%

CXCR5

PD-1

dsRed (xfer)

GFP (Foxp3)
0 103104105

0103104105

0

103

104

105

0

103

Late GCs

Immunize:
NP-OVA-alum
10 μg footpad

Day: -1 19/20

Transfer: (1.0-1.4 x 10^7 ) MACS + FACS double-sorted
dsRed+ GFP- naïve, polyclonal CD4+ T cells
from dsRed-tgFoxp3GFP donors

FDC labeling:
anti-CD35-AF633 i.v.

Image

0 9 10

Recipient:
P25 TCR-tg

A

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