Science - USA (2021-07-16)

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individual GCs as they approached their dis-
solution phase (Fig. 2, A and B; fig. S2A; and
movie S1). Longitudinal imaging showed that
the surge in Foxp3+T cells most often takes
place immediately before the onset of GC con-
traction (Fig. 2, C to F; and fig. S2, A and B).
AligningGCsintimebythepeakofthenum-
ber of Foxp3+T cells revealed a doubling in the
number of Foxp3+T cells within the 24-hour
period preceding the peak, which was directly
followed by an almost 50% decrease in GC
volume over the next 24 hours (Fig. 2, E and
F). Thus, the surge in GC-localized Foxp3+
T cells immediately precedes GC contraction,
consistent with these cells playing a direct role
in this process.


Foxp3+T cells engage in prolonged interactions
with B cells in end-stage GCs
Given the evidence that direct contacts between
Tregsand GC B cells may contribute to the reg-
ulation of humoral responses ( 23 ), we investi-
gated the interactions between B cells and
Foxp3+T cells at different times during GC
evolution by intravital multiphoton micros-
copy. To establish that our system is capable of
discerning Treg–B cell interactions, we first im-
aged cells at day 2 postimmunization, when
effector T and B cell contacts taking place at
the T cell zone–B cell follicle (T:B) border are
long-lasting ( 24 ). We transferred CFP+B1-8hi
B cells (CFP, cyan fluorescent protein) along with
RFP+OT-II T cells also expressing the fluores-

cent Ca2+reporter GCaMP3 intoFoxp3GFPhosts,
and imaged pLNs at 2 days after footpad im-
munization with NP-OVA in alum. In this set-
ting, Foxp3+T cells and B1-8hiB cells engaged
in clearly identifiable interactions, which,
although generally briefer than those seen
between B cells and helper T cells, could oc-
casionally last for 10 min or longer (Fig. 3, A
and B; and Movie 1). These interactions fell
into two distinct modalities: Either multiple
Foxp3+T cells“swarmed”over specific B cells,
or B1-8hiB cells dragged a single Foxp3+T cell
behind them, as previously described for inter-
actions with helper T cells (Fig. 3A and Movie 1)
( 24 , 25 ). Thus, prolonged direct interactions be-
tween B cells and Foxp3+T cells do take place

Jacobsenet al.,Science 373 , eabe5146 (2021) 16 July 2021 2 of 13


A

Host:
Foxp3GFP/Y

Transfer:
5x10^5 NP+ B1-8hi
10% CFP+/90% CFP–

Boost:
NP-OVA 25 μg footpad

Transfer:
103 OT-II
dsRed

Prime:
OVA-alum
50 μg i.p.

Image

Day: -16 -14 -1 0 6 10 14/15

PAGFP-tg
Foxp3RFP

Photoactivate, FACS

Day: -14 0 10 14/15

Prime:
OVA-alum
50 μg i.p.

Boost:
NP-OVA
25 μg footpad

C P = 0.004
P < 0.0001

0

20

40

60

Foxp3

+ cells/(100 μm)

3

Days post-boost

6 10 14/15

0

100

200

300

P = 0.027
P = 0.025

Foxp3

+ cells/GC

Days post-boost

6 10 14/15

0

5

10

15

GC volume μm

3 (x10

6 )

Days post-boost

6 10 14/15

P = 0.023

B Day 10 post-boost^ Foxp3 OT-II B1-8hi

i.

ii.

iii.

iii.

i.

ii.

iii.
Day 14 post-boost Foxp3 OT-II B1-8hi

i.

i.
ii.

ii.

iii.

iii.

D Photoactivation of T cells in prime-boost GCsE GC T cells (prime-boost) F

Pre-photoactivation Post-photoactivation

PE/Anti-PE (FDCs) Photoactivation

Day 10
post-boost^0

20

40

60

80

Foxp3

+

(% of photoactivated)

0

20

40

60

80

100

Day 10
Day 14/15

P = 0.0016

Foxp3+ Foxp3-

CXCR5

+/PD-1

hi

(% of photoactivated)

Days post-boost

0

1

2

4
3

5

GC size (% of B cells)
Days post-boost

10

P < 0.0001

14/15^10 14/15

P = 0.039

Day 15
post-boost

Foxp3-RFP

PAGFP (activated)

CXCR5

Foxp3- Foxp3+
105
104
103

0
0 103104105

105
104
103

0
0 103 104105

105
104
103

0
0 103 104105

Gated on CD4+

21 74 39

PD-1

105
104
103
0
0 103104105

105
104
103
0

0103 104105

105
104
103
0

0103 104105

42

74 63

Fig. 1. Kinetics of Foxp3-expressing T cells throughout the course of the GC
reaction.(A) Experimental setup. (B) Multiphoton image of single optical slices of
GCs at 10 and 14 days postboost, followed by computational rendering of the entire
GC volume. Foxp3+cells are marked with dashed circles (images; yellow circles
indicate cells magnified in the insets to the right) or green spheres (renderings).
Scale bars: 30mm (large panels) and 10mm (insets). (C) Quantification of data
in (B). Each symbol corresponds to one GC. Two to three GCs were counted per
mouse in at least three independent experiments per time point with two mice per


group. (D) Experimental setup (top). GCs were identified by labeling follicular
dendritic cells (FDCs) with anti-phycoerythrin (PE)–PE immune complexes prior to
imaging and photoactivation (bottom). Scale bar: 100mm. i.p., intraperitoneally.
(EandF) Frequency and phenotype of photoactivated RFP+and RFP–CD4+T cells
in single GCs. Each symbol represents one GC. Data are pooled from at least
four independent experiments with the mean value indicated. Data for GC size in (F)
are from two independent experiments, each symbol representing one mouse. All
Pvalues are for paired StudentÕsttest, performed only for the comparisons indicated.

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