Article

The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood

1] Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
Nature Immunology (Impact Factor: 20). 12/2012; 14(2). DOI: 10.1038/ni.2496
Source: PubMed

ABSTRACT

CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (T(FR) cells) inhibit humoral immunity mediated by CD4(+)CXCR5(+)Foxp3(-) follicular helper T cells (T(FH) cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of T(FR) cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of T(FR) cells in the lymph nodes and that those T(FR) cells had enhanced suppressive ability. We also found substantial populations of T(FR) cells in mouse blood and demonstrated that T(FR) cells in the blood homed to lymph nodes and potently inhibited T(FH) cells in vivo. T(FR) cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.

1 5 2 VOLUME 14 NUMBER 2 FEBRUARY 2013 nature immunology
A R T I C L E S
Follicular helper T cells (T
FH
cells) are a subset of CD4
+
T cells that are
essential for helping cognate B cells form and maintain the germinal
center (GC) reaction and for the development of humoral immune
responses. These cells are universally defined by expression of the
chemokine receptor CXCR5, which directs them to B cell folli-
cles via gradients of the chemokine CXCL13 (ref. 1). T
FH
cells also
express the transcription factor Bcl-6 and have high expression of the
costimulatory receptor ICOS. Both Bcl-6 and ICOS are critical for the
differentiation and maintenance of T
FH
cells
1−4
. In addition, T
FH
cells
secrete large amounts of interleukin 21 (IL-21), which aids in the for-
mation of GCs, isotype switching and the formation of plasma cells
5
.
In humans and mice, functionally similar T
FH
cells can be found in
secondary lymphoid organs. CXCR5
+
T
FH
cells are also present in
peripheral blood and are found in greater abundance in people with
autoantibodies, including patients with systemic lupus erythematosus,
myasthenia gravis or juvenile dermatomyositis. However, the function
of circulating T
FH
cells has remained unclear
6–9
.
T
FH
cells also have high expression of the inhibitory receptor PD-1
(CD279). Signaling through PD-1 attenuates signaling from the
T cell antigen receptor (TCR) and inhibits the population expansion,
cytokine production and cytolytic function of T cells. In addition,
PD-1 promotes the development of induced regulatory T cells (T
reg
cells) from naive lymphocytes
10–14
. PD-1 has two ligands: PD-L1
(B7-H1) and PD-L2 (B7-DC). PD-L1 has wider expression than does
PD-L2, but both PD-L1 and PD-L2 can be expressed on GC B cells
and dendritic cells
15
. Perturbation studies have suggested critical
roles for this pathway in regulating humoral immune responses.
However, there are conflicting reports about the function of the
PD-1 pathway in controlling humoral immunity. When interactions
between PD-1 and its ligand(s) are prevented, some studies have
found attenuated humoral responses
16–18
, whereas others have
observed heightened humoral responses
19,20
.
PD-1 also is found on the CD4
+
CXCR5
+
subset of cells calledfol-
licular regulatory T cells (T
FR
cells), which express the transcrip-
tion factors Foxp3, Bcl-6 and Blimp-1 and function to inhibit the GC
response
21–23
. These cells originate from natural T
reg
cells but have
expression of ICOS, CXCR5 and PD-1 similar in amount to that of
T
FH
cells. As ICOS, CXCR5 and PD-1 have been widely used to iden-
tify and purify T
FH
cells’, it seems likely that the inability to define
clear functions for PD-1 in GC responses derives from experimental
systems that contain mixtures of stimulatory T
FH
cells and inhibitory
T
FR
cells. Separate analyses of the function of PD-1 on T
FH
cells and
T
FR
cells are needed to elucidate how PD-1 controls humoral immu-
nity and to gain insight into the individual roles of T
FR
cells and T
FH
cells in the regulation of antibody production.
Here we found that PD-1–PD-L1 interactions inhibited the number
of T
FR
cells, but not of T
FH
cells, in the lymph nodes. PD-1-deficient
mice had more T
FR
cells in the lymph nodes than did wild-type mice.
PD-1-deficient T
FR
cells in the lymph nodes had an enhanced ability
to suppress both the activation of naive T cells and antibody produc-
tion in vitro. In addition, we found that T
FR
cells were present in
the peripheral blood of mice and that these circulating cells potently
regulated humoral immune responses in vivo. Through the use
of transfer approaches, we found that T
FH
cells in the blood pro-
moted antibody production, whereas T
FR
cells in the blood strongly
inhibited antibody production in vivo. We further found that the
PD-1 pathway inhibited the function of T
FR
cells in the blood and
that PD-1-deficient T
FR
cells in the blood had enhanced suppressive
ability in vivo. Together our studies identify a previously unknown
immunoregulatory role for the PD-1–PD-L1 pathway in limiting the
1
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
2
Department of Medicine, Beth Israel Deaconess Medical
Center, Boston, Massachusetts, USA.
3
Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA. Correspondence should be addressed
to A.H.S. (arlene_sharpe@hms.harvard.edu).
Received 1 October; accepted 15 November; published online 16 December 2012; doi:10.1038/ni.2496
The receptor PD-1 controls follicular regulatory
T cells in the lymph nodes and blood
Peter T Sage
1,2
, Loise M Francisco
1,3
, Christopher V Carman
2
& Arlene H Sharpe
1,3
CD4
+
CXCR5
+
Foxp3
+
follicular regulatory T cells (T
FR
cells) inhibit humoral immunity mediated by CD4
+
CXCR5
+
Foxp3
follicular
helper T cells (T
FH
cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of
T
FR
cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of T
FR
cells in
the lymph nodes and that those T
FR
cells had enhanced suppressive ability. We also found substantial populations of T
FR
cells
in mouse blood and demonstrated that T
FR
cells in the blood homed to lymph nodes and potently inhibited T
FH
cells in vivo.
T
FR
cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-
L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile
inconsistencies surrounding the role of this pathway in humoral immunity.
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© 2013 Nature America, Inc. All rights reserved.
Page 1
nature immunology VOLUME 14 NUMBER 2 FEBRUARY 2013 1 5 3
A R T I C L E S
differentiation and function of T
FR
cells and further demonstrate the
dynamic control of humoral immune responses through the migra-
tion of T
FR
cells from the circulation into the lymph nodes to control
antibody production in vivo.
RESULTS
PD-1 controls T
FR
cells
To analyze the role of PD-1 in controlling T
FR
cells, we first assessed
PD-1 expression on CD4
+
T cell subsets in the draining lymph nodes
of wild-type C57BL/6 mice immunized subcutaneously with myelin
oligodendrocyte glycoprotein peptide (amino acids 35–55) emulsi-
fied in complete Freund’s adjuvant (collectively called ‘MOG’ here),
an immunization that breaks tolerance and also results in the effective
generation of T
FH
cells
24
. We defined T
FR
cells as CD4
+
ICOS
+
CXCR5
+
Foxp3
+
CD19
, a gating strategy that separated T
FR
cells from
CD4
+
ICOS
+
CXCR5
+
Foxp3
CD19
T
FH
cells, the cell type previously
thought to solely constitute the CD4
+
CXCR5
+
gate (Fig. 1a). T
FH
cells had higher expression of PD-1 protein than did ICOS
+
CXCR5
effector-like cells or ICOS
CXCR5
naive cells (called ‘naive cells’
here) in the draining lymph nodes on day 7 after immunization
(
Fig. 1b). Notably, T
FR
cells had even higher expression of PD-1 protein
than did any other CD4
+
T cell subset examined, including T
FH
cells (
Fig. 1b).
To determine the functional importance of PD-1 expression on
T
FR
cells, we immunized wild-type and PD-1-deficient (Pdcd1
−/−
)
mice and analyzed T
FR
cells 7 d later. The frequency of T
FR
cells in
the CD4
+
Foxp3
+
gate was about 4% in wild-type lymph nodes and
<1% of all CD4
+
T cells. In contrast, the frequency of T
FR
cells in
Pdcd1
−/−
mice was about 10% of the CD4
+
Foxp3
+
gate and >2% of
all CD4
+
T cells (Fig. 1c,d). Because the total number of CD4
+
T cells
was typically about twofold higher in the PD-1-deficient lymph nodes
than in wild-type lymph nodes, a twofold-greater frequency of T
FR
cells indicated an approximately fourfold-greater absolute number
of T
FR
cells (data not shown). When determined as a percentage of
all CD4
+
ICOS
+
CXCR5
+
cells (and therefore as the percentage of
CD4
+
T cells that could respond to CXCL13 and migrate to the B cell
zone), Pdcd1
−/−
T
FR
cells constituted 50% of that population, whereas
wild-type T
FR
cells constituted only about 20% (Fig. 1d). We also
observed a much greater frequency of T
FR
cells in Pdcd1
−/−
mice when
we immunized the mice with other classic B cell antigens, such as the
hapten NP (4-hydroxy-3-nitrophenylacetyl) conjugated to ovalbumin
(NP-OVA; Supplementary Fig. 1). We did not find a significant dif-
ference in the frequency of Foxp3
T
FH
cells (called ‘T
FH
cells’ here)
when determined as a percentage of all CD4
+
T cells in wild-type and
Pdcd1
−/−
mice on day 7 after immunization with MOG (Fig. 1e) or
NP-OVA (Supplementary Fig. 1).
As PD-1 can be expressed by many types of hematopoietic cells,
including T cells, B cells, macrophages and some dendritic cells
15
,
we next investigated whether PD-1 regulates T
FR
cells directly by
controlling their generation from Foxp3
+
T
reg
cells. To track the
fate of Foxp3
+
cells after transfer into wild-type mouse recipients,
we used antigen-specific Foxp3
+
T cells from 2D2 mice, which have
transgenic expression of a MOG-specific TCR, for these studies.
We sorted Foxp3
+
T
reg
cells from wild-type 2D2 or Pdcd1
−/−
2D2
mice that expressed a Foxp3–green fluorescent protein (GFP) reporter
(Foxp3-GFP reporter mice), then transferred 2 × 10
5
wild-type or
Pdcd1
−/−
2D2 CD4
+
CXCR5
Foxp3
+
cells into wild-type recipient
mice. We immunized the recipients with MOG and analyzed cells in
the draining lymph nodes 7 d later. A greater frequency (Fig. 1f,g)
and absolute number (Fig. 1h) of Pdcd1
−/−
2D2 T
reg
cells than wild-
type 2D2 T
reg
cells upregulated CXCR5 and thus differentiated into
T
FR
cells. The greater frequency of Pdcd1
−/−
T
FR
cells in the immu-
nized recipients of cell transfer was similar to but slightly less than the
greater frequency of T
FR
cells in immunized mice lacking PD-1 on all
cells (Fig. 1d,g). These results demonstrated that PD-1 controlled the
differentiation of Foxp3
+
T
reg
cells into T
FR
cells.
c
WT
Pdcd1
–/–
4.63
10.3
ICOS
CD4
CXCR5
Foxp3
10
20
30
40
50
***
1
2
3
NS
e
WT
Pdcd1
–/–
WT
Pdcd1
–/–
d
2
WT
Pdcd1
–/–
1
2
3
***
WT
Pdcd1
–/–
4
6
8
10
***
g
WT
Pdcd1
–/–
2
4
6
8
10
12
*
T
FR
cells
(% of Foxp3
+
)
WT
Pdcd1
–/–
100
200
300
400
500
**
T
FR
(per LN)
hf
6.04
10.2
WT
Gated on CD4
+
Foxp3
+
Pdcd1
–/–
ICOS
CXCR5
T
FR
(% of Foxp3
+
)
T
FR
(% of CD4
+
)
T
FR
(% of CD4
+
ICOS
+
CXCR5
+
)
T
FH
(% of CD4
+
)
a
PD-1 (MFI)
200
Naive
ICOS
+
T
FH
T
FR
400
600
800
1,000
1,200
5.26
ICOS
+
Naive
21.3
T
FH
T
FR
78.7
CD4 ICOS CD4
CD19
CXCR5
Foxp3
b
PD-1
Frequency
ICOS
+
Naive
T
FR
T
FH
Figure 1 PD-1 signaling in Foxp3 T
reg
cells limits generation of T
FR
cells. (a) Quantification of naive (CD4
+
ICOS
CXCR5
CD19
) cells and ICOS
+
(CD4
+
ICOS
+
CXCR5
CD19
) cells (middle) and T
FR
(CD4
+
Foxp3
+
ICOS
+
CXCR5
+
CD19
) cells and T
FH
(CD4
+
Foxp3
ICOS
+
CXCR5
+
CD19
) cells (right)
in draining lymph nodes of wild-type mice 7 d after immunization with MOG. Numbers in outlined areas indicate percent cells in each throughout.
(b) PD-1 expression on wild-type naive, ICOS
+
, T
FR
and T
FH
cells gated as in a, assessed by flow cytometry. (c) Gating of T
FR
cells among total Foxp3
+
cells in wild-type and Pdcd1
−/−
mice 7 d after immunization with MOG. (d) Quantification of wild-type and Pdcd1
−/−
T
FR
cells from the inguinal lymph
nodes, gated in c, presented as frequency among CD4
+
Foxp3
+
cells (left), total CD4
+
T cells (middle) or in the CD4
+
ICOS
+
CXCR5
+
CD19
gate (right).
(e) Quantification of wild-type and Pdcd1
−/−
T
FH
cells from the inguinal lymph nodes, as a percentage of CD4
+
T cells. (f) Gating of T
FR
cells from the
lymph nodes of wild-type recipients given wild-type 2D2 and Pdcd1
−/−
2D2 CD4
+
Foxp3
+
CXCR5
T
reg
cells (2 × 10
5
), followed by immunization with
MOG and analysis 7 d later. (g,h) Quantification of T
FR
cells from transfer experiments as in f, presented as the frequency of Foxp3
+
cells on day 7 after
immunization (g) or total cells per lymph node (h). NS, not significant; *P < 0.05, **P < 0.005 and ***P < 0.0005 (unpaired Student’s t-test). Data are
representative of at least two experiments with at least five mice per group (error bars, s.e.m.).
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Page 2
1 5 4 VOLUME 14 NUMBER 2 FEBRUARY 2013 nature immunology
A R T I C L E S
As CD25 (the α-chain of the receptor for IL-2) is often used as a
marker for T
reg
cells, we next compared CD25 expression on wild-type
and Pdcd1
−/−
T
FR
cells immediately after isolation (Fig. 2a). Pdcd1
−/−
T
FR
cells had lower expression of CD25 than did wild-type T
FR
cells
(Fig. 2b). The attenuated CD25 expression in PD-1-deficient T
FR
cells
was probably not due to less activation because expression of the early
activation marker CD69 was almost completely identical on wild-
type and Pdcd1
−/−
T
FR
cells (Fig. 2c). To compare the proportion of
wild-type and Pdcd1
−/−
T
FR
cells that were proliferating on day 7 after
immunization with MOG, we examined expression of Ki67, a marker
widely used to identify cells that are actively dividing. Wild-type
CXCR5
ICOS
+
effector cells, T
FH
cells and T
FR
cells had high expres-
sion of Ki67. In contrast, wild-type CXCR5
ICOS
‘naive’ cells, which
lacked expression of CD69 and CD25, had no Ki67 staining, consistent
with their designation as naive (Fig. 2d). Wild-type T
FR
cells had much
higher expression of Ki67 than did Pdcd1
−/−
T
FR
cells (Fig. 2d), which
suggested that the greater abundance of T
FR
cells in PD-1-deficient
mice reflected more differentiation, not maintenance, of T
FR
cells.
Ki67 expression was similarly higher in wild-type ICOS
+
effector
cells and T
FH
cells than in Pdcd1
−/−
ICOS
+
CXCR5
effector cells and
T
FH
cells. This indicated a lower amount of cycling of Pdcd1
−/−
effec-
tor cells at 7 d after immunization. The expression of other T
reg
cell
markers, such as CD103 and GITR, was not altered on T
FR
cells in
PD-1-deficient mice (Supplementary Fig. 2). Additionally, there was
low but substantial expression of PD-L1 on wild-type and Pdcd1
−/−
T
FR
cells (Supplementary Fig. 2). Together these data indicated that
PD-1 was important in regulating the number of T
FR
cells in vivo.
PD-1-deficient T
FR
cells can home to GCs
We next compared the ability of wild-type and PD-1-deficient T
FR
cells to enter the GC to inhibit the GC response. First we evaluated GC
formation in lymph node sections collected 7 d after immunization
with MOG. We identified GCs by the binding of peanut agglutinin
and positive staining for the GC marker GL7 and lack of staining for
immunoglobulin D (IgD; Fig. 3a). We determined that these GCs
were active on the basis of their robust expression of Ki67 (Fig. 3b).
Similar to published reports
21,22
, we found CD4
+
Foxp3
+
T
FR
cells
in the GCs of immunized mice (Fig. 3c). The Foxp3 protein in the
T
FR
cells was mostly nuclear, given its localization together with the
DNA-intercalating dye DAPI (Fig. 3d).
We then investigated whether the phenotypically distinct PD-1-
deficient T
FR
cells were able to migrate to GCs similarly to wild-type
T
FR
cells because blockade of PD-1 can prolong the TCR ‘stop signal’
and diminish T cell migration
25
. We immunized wild-type and PD-1-
deficient mice with MOG and analyzed lymph node sections 7 d later
for expression of IgD, GL7 and Foxp3 (Fig. 3e). Although the GC area
(Fig. 3f) and number of GCs per lymph node (data not shown) were
equivalent in wild-type and Pdcd1
−/−
mice, there were slightly more
Foxp3
+
cells (and therefore T
FR
cells) located within the GC borders
in Pdcd1
−/−
mice than in wild-type mice (Fig. 3g). However, as this
was proportional to the greater number of T
FR
cells in PD-1-deficient
mice determined by flow cytometry, these data demonstrated that
PD-1-deficient T
FR
cells were not defective in homing to GCs and
were able to enter the GC like wild-type T
FR
cells.
The location of Foxp3
+
T
FR
cells in the GC did not differ signifi-
cantly for wild-type and Pdcd1
−/−
mice (Fig. 3h). In both wild-type
and Pdcd1
−/−
mice, the Foxp3
+
cells tended to reside near the GC bor-
der, with more than half of the nuclei of Foxp3
+
cells being positioned
within 10 µm of the border. Furthermore, when we quantified CXCR5
fluorescence by flow cytometry, we found similar CXCR5 expression
on wild-type and Pdcd1
−/−
T
FR
cells (Fig. 3i), which indicated a simi-
lar potential for these cells to respond to chemokine cues to migrate
to GCs. Together these data indicated that the lymph nodes of immu-
nized Pdcd1
−/−
mice had more T
FR
cells and that these Pdcd1
−/−
T
FR
cells were able to migrate into GCs to regulate B cell responses.
PD-1-deficient T
FR
cells more potently inhibit antibody
We next assessed the function of T
FR
cells from wild-type and
Pdcd1
−/−
mice. T
FR
cells have higher expression of the T
reg
cell–
associated receptor GITR on the surface than do T
FH
cells, which
allowed us to separate T
FH
cells and T
FR
cells in a similar manner to
that of intracellular staining for Foxp3 (Fig. 4a). For functional stud-
ies, we sorted T
FR
cells from immunized mice by defining the lymph
node CD4
+
ICOS
+
CXCR5
+
CD19
GITR
+
population as T
FR
cells
and the CD4
+
ICOS
+
CXCR5
+
CD19
GITR
population as T
FH
cells
(
Fig. 4b). Sorting in this way resulted in a considerable abundance of
Foxp3 mRNA in the GITR
+
(T
FR
) population but essentially no Foxp3
mRNA in the GITR
(T
FH
) population (Fig. 4b), which confirmed the
utility of this gating strategy for the isolation of T
FR
cells and T
FH
cells
for functional assays. Furthermore, we were able to use this sorting
strategy to compare wild-type and PD-1-deficient T
FR
cells, as GITR
expression was identical on wild-type and PD-1-deficient T
FR
cells
(Supplementary Fig. 3).
T
FR
cells have high expression of Blimp-1 and moderate expres-
sion of Bcl-6 (ref. 21). Bcl-6 and Blimp-1 reciprocally modulate each
other
2
; inhibition of Blimp-1 by Bcl-6 is essential for maintenance of
a
4.66
15.9
ICOS
CD4
T
FH
ICOS
+
Naive
T
FR
83.5
Gated on
CD4
+
CD19
CXCR5
Foxp3
b
CD25
WT T
FR
Pdcd1
–/–
T
FR
Frequency
1
2
WT
Pdcd1
–/–
WT
Pdcd1
–/–
WT
Pdcd1
–/–
WT
Pdcd1
–/–
3
***
***
CD25 (10
3
MFI)
T
FR
T
FH
ICOS
+
Naive
c
CD69
WT T
FR
Pdcd1
–/–
T
FR
Frequency
1
2
3
**
***
NS
WT
Pdcd1
–/–
WT
Pdcd1
–/–
WT
Pdcd1
–/–
WT
Pdcd1
–/–
T
FR
T
FH
ICOS
+
Naive
CD69 (10
3
MFI)
d
Ki67
WT T
FR
Pdcd1
–/–
T
FR
Frequency
T
FR
T
FH
ICOS
+
Naive
20
40
60
80
100
***
**
*
WT
Pdcd1
–/–
WT
Pdcd1
–/–
WT
Pdcd1
–/–
WT
Pdcd1
–/–
Ki67
hi
(%)
Figure 2 PD-1-deficient T
FR
cells have altered expression of activation markers. (a) Gating strategy
for the identification of T
FR
cells in draining lymph nodes collected from wild-type or Pdcd1
−/−
mice 7 d after immunization with MOG. (b,c) Expression of CD25 (b) and CD69 (c) on wild-type
and Pdcd1
−/−
CD4
+
subsets gated as in a, on T
FR
cells (left) and as mean fluorescence intensity
(MFI; right). (d) Intracellular staining of Ki67 in wild-type and Pdcd1
−/−
CD4
+
subsets gated as
in a, on T
FR
cells (left) and as mean fluorescence intensity of Ki67
hi
cells (right), defined as the
highest intensity peak on wild-type T
FR
cells (horizontal line at left). *P < 0.05, **P < 0.005 and
***P < 0.0005 (unpaired Students t-test). Data are representative of at least two independent
experiments (mean and s.e.m. of five mice per group).
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nature immunology VOLUME 14 NUMBER 2 FEBRUARY 2013 1 5 5
A R T I C L E S
the T
FH
phenotype, whereas Blimp-1 is important in the homeostasis
of T
reg
cells in general
26,27
. As relative expression of Bcl-6 and Blimp-
1 determines the function of T
FH
subsets, we compared intracellular
Bcl-6 expression in wild-type T
FR
cells with that in Pdcd1
−/−
T
FR
cells
by flow cytometry. Although T
FR
cells had lower expression of Bcl-6
than did T
FH
cells, wild-type and Pdcd1
−/−
T
FR
cells had similar
amounts of Bcl-6 (Fig. 4c). We next compared expression of the gene
encoding Blimp-1 (Prdm1) in wild-type T
FR
cells with that in Pdcd1
−/−
T
FR
cells. We did not find any consistent difference between wild-
type and Pdcd1
−/−
T
FR
cells in Prdm1 mRNA expression (Fig. 4d). As
Foxp3 can directly interact with and negatively regulate the function
of the transcription factor RORγt
28
, we also examined the expression
of Rorc (which encodes RORγt) in wild-type and Pdcd1
−/−
T
FR
cells.
T
FR
cells had lower expression of Rorc mRNA than did T
FH
cells, but
Rorc expression was higher in Pdcd1
−/−
T
FR
cells than in wild-type T
FR
cells (Fig. 4e). In addition, we compared expression of the transcrip-
tion factor IRF4 in wild-type T
FR
cells with that in Pdcd1
−/−
T
FR
cells,
as Blimp-1 and IRF4 synergistically control the differentiation and
effector functions of T
reg
cells
26
. We found higher expression of Irf4
mRNA in Pdcd1
−/−
T
FR
cells than in wild-type T
FR
cells (Fig. 4f).
IRF4 is essential for the suppressive ability of T
reg
cells
26
. To
determine if higher expression of Irf4 mRNA in
Pdcd1
−/−
T
FR
cells
resulted in more suppression of the proliferation of T cells, we set up
an in vitro suppression assay in which we cultured sorted wild-type
CD4
+
CD62L
+
Foxp3
responder T cells (from unimmunized mice)
labeled with the cytosolic dye CFSE, wild-type GL7
B cells from
MOG-immunized wild-type mice and T
FR
cells sorted from MOG-
immunized wild-type or Pdcd1
−/−
mice, together with antibody to
CD3 (anti-CD3) and anti-IgM. The responder T cells had substantial
upregulation of the expression of CD69 after 3 d of culture with wild-
type B cells. However, when we added wild-type T
FR
cells (at a ratio
of 1:1:1 with responder T cells and B cells as described above), the
CD69 expression on the responder T cells was much lower (Fig. 4g),
consistent with the function of T
FR
cells in suppressing T cell activa-
tion. CD69 upregulation was inhibited to an even greater extent in
responder T cells cultured with Pdcd1
−/−
T
FR
cells. Moreover, Pdcd1
−/−
T
FR
cells attenuated the proliferation of responder T cells, in con-
trast to wild-type T
FR
cells, which did not inhibit the proliferation of
responder T cells during the 3-day culture period (Fig. 4h).
Although T
FR
cells are thought to inhibit the GC response in vivo,
it is unclear whether T
FR
cells directly inhibit the differentiation of
T cells, the function of T
FH
cells, the activation of B cells or all three.
To assess the ability of T
FR
cells to suppress antibody production by
B cells in vitro, we cultured wild-type GL7
B cells with wild-type
T
FH
cells for 6 d in the presence or absence of T
FR
cells (all from
MOG-immunized mice), anti-IgM and anti-CD3. Wild-type B cells
produced large amounts of IgG when cultured with wild-type T
FH
cells plus anti-IgM and anti-CD3 (Fig. 4i). No significant IgG was
produced when we used naive CD4
+
T cells in these experiments
(Fig. 4j). When we added T
FR
cells to the wells along with T
FH
cells,
almost no IgG was produced. The T
FR
cell–mediated suppression was
not due to the sequestering of anti-CD3 because there was equally
good suppression at the two doses of anti-CD3 tested (Fig. 4i) and
anti-CD3 was still present on the surface of the T
FH
cells at the end
of the suppression assay (Supplementary Fig. 4). Pdcd1
−/−
T
FR
cells
suppressed IgG production more than wild-type T
FR
cells did at ratios
Figure 3 PD-1-deficient T
FR
cells are able
to home to GCs. (a) Microscopy of draining
lymph nodes from wild-type mice 7 d after
immunization with MOG, stained for GL7
(green), binding of peanut agglutinin (PNA; red)
and IgD (blue). GCs (white dashed lines)
are PNA
+
GL7
+
IgD
. (b) Microscopy of GC
sections as in a, stained for Ki67 (blue),
Foxp3 (red) and GL7 (green). (c) Microscopy
of the colocalization of CD4 (blue) and Foxp3
(red) and GL7 (green) in sections as in a;
outlined area (far right) indicates area enlarged
below, with CD4
+
staining on Foxp3
+
cells.
(d) Microscopy of the localization of Foxp3
in the nucleus in sections stained with DAPI
(blue) and for Foxp3 (red) and GL7 (green)
in sections as in a; outlined area (far right)
indicates area enlarged below, showing Foxp3
in DAPI
+
nuclei. (e) Microscopy of Foxp3
+
T
FR
cells in GCs of draining lymph nodes obtained
from wild-type and Pdcd1
−/−
mice 7 d after
immunization with MOG. (f) Area of GCs as in e,
calculated as the area in the dashed lines
in e (mean area per lymph node). (g) Foxp3
+
cells in GCs as in e (mean per GC). (h) Distance
from Foxp3
+
cells to the border of GCs as in e
(dashed line in e). Each symbol represents an
individual FoxP3
+
cell; small horizontal lines
indicate the mean. (i) CXCR5 expression on
wild-type and Pdcd1
−/−
T
FH
and T
FR
cells
7 d after MOG immunization, assessed by
flow cytometry. Original magnification, ×400
(a,b,e and main image in c,d), ×2,480
(c, enlargement) or ×2320 (d, enlargement).
NS, not significant; *P < 0.05, **P < 0.005, ***P < 0.0005 (unpaired Student’s t-test). Data are representative of three experiments (a
d,i; mean
and s.e.m. of five mice per group in i) or two experiments (eh; mean and s.e.m. of ten lymph nodes in fh).
a
IgD PNA GL7 Merge
Ki67 Foxp3 GL7 Merge
CD4 Foxp3 GL7 Merge
DAPI Foxp3 GL7 Merge
WT
IgD
GL7
Foxp3
Pdcd1
–/–
e
b
c
d
f g
NS
10,000
GC area (µm
2
)
8,000
6,000
4,000
2,000
WT
Pdcd1
–/–
10
*
T
FR
per GC
WT
Pdcd1
–/–
8
6
4
2
h i
T
FR
distance to
GC border (µm)
NS
70
WT
Pdcd1
–/–
60
50
40
30
20
10
T
FH
T
FR
CXCR5 (10
3
MFI)
14
12
10
8
6
4
2
NS
**
***
*
WT
Pdcd1
–/–
WT
Pdcd1
–/–
npg
© 2013 Nature America, Inc. All rights reserved.
Page 4
1 5 6 VOLUME 14 NUMBER 2 FEBRUARY 2013 nature immunology
A R T I C L E S
of T
FR
cells to T
FH
cells of 1:1 (Fig. 4j) and 1:5 (Fig. 4k), with PD-1-
deficient T
FR
cells resulting in 50% lower IgG production than that of
wild-type T
FR
cells. Together these data demonstrated not only that
Pdcd1
−/−
mice had more T
FR
cells but also that those Pdcd1
−/−
T
FR
cells had greater suppressive capacity.
PD-1 controls T
FR
cells in the blood
One possible explanation for the greater abundance of T
FR
cells in
lymph nodes of immunized PD-1-deficient mice might be that Pdcd1
−/−
T
FR
cells were unable to exit the lymph node. Functional T
FH
cells can
be found in the blood of humans as well as mice
6,7,9
, but whether
T
FR
cells circulate in the blood of humans or mice is not yet known.
We found a distinct population of T
FH
cells, as well as a smaller popu-
lation of T
FR
cells, in the blood of wild-type mice immunized with
MOG (Fig. 5a,b). When we assessed the kinetics of the population
expansion of T
FH
cells and T
FR
cells in the lymph node and blood of
mice after MOG immunization, we found that the frequency of T
FR
and T
FH
cells increased in the draining lymph nodes of immunized
wild-type mice over a 10-day period, and that T
FH
cells, but not T
FR
cells, increased substantially (by percentage) in the blood over this
time (Fig. 5b). Thus, without antigenic stimulus, the ratio of T
FR
cells to T
FH
cells in the blood was fairly high (sometimes >1:1), but
after the addition of a stimulus, the population expansion of T
FH
cells in the blood was greater than that of T
FR
cells in the blood, so
that the ratio of T
FR
cells to T
FH
cells was about 1:5. To investigate
whether wild-type T
FH
cells and T
FR
cells in the blood were quiescent
or actively in the cell cycle, we assessed Ki67 expression in T
FH
cells
and T
FR
cells from the draining lymph nodes and blood at 7 d after
immunization. T
FH
cells from the draining lymph nodes had higher
Ki67 expression than did those in the blood (Fig. 5c). T
FH
cells and
T
FR
cells in the blood and T
FR
cells from the draining lymph nodes
had similar expression of Ki67.
Next we investigated whether T
FR
cells in the blood were inhibited
to the same degree by PD-1 signaling as were T
FR
cells from the lymph
nodes. We immunized wild-type and Pdcd1
−/−
mice with MOG and
7 d later analyzed T
FH
cells and T
FR
cells from the blood. In wild-type
mice, ~2–3% of CD4
+
Foxp3
CD19
cells in the blood were T
FH
cells,
but in the Pdcd1
−/−
mice, ~4–5% were T
FH
cells (Fig. 5d). This greater
frequency of Pdcd1
−/−
T
FH
cells in blood was in contrast to results
obtained for the lymph nodes, where Pdcd1
−/−
mice had a frequency
of T
FH
cells similar to, if not less than, that of wild-type mice (Fig. 1e).
Notably, T
FR
cells constituted ~3% of all Foxp3
+
cells in the blood of
wild-type mice but >7% of Foxp3
+
cells in the blood of Pdcd1
−/−
mice
(Fig. 5d,e). The greater frequency of Foxp3
+
cells seemed to be spe-
cific to the subset of T
FR
cells in the blood, as the frequency of Foxp3
+
cells in the ICOS
+
CXCR5
(ICOS
+
) and ICOS
CXCR5
naive cell
gates was not greater in Pdcd1
−/−
mice (Fig. 5f). Together these data
indicated that both T
FR
cells and T
FH
cells were present in the blood
of mice and that both subsets were repressed by PD-1 signaling.
To investigate whether T
FH
and T
FR
cells in the blood had a central
memory phenotype, we analyzed surface expression of CD62L and
CD44. About 60% of wild-type and Pdcd1
−/−
T
FR
cells in the blood
had high expression of CD62L (Supplementary Fig. 5). This was in
contrast to the finding that >90% of ICOS
CXCR5
naive cells had
high expression of CD62L. Pdcd1
−/−
T
FH
cells had lower expression
of CD62L than did wild-type T
FH
cells. All wild-type and Pdcd1
−/−
T
FR
cells in the blood had high expression of CD44, but Pdcd1
−/−
T
FR
cells in the blood had slightly lower surface expression than did
T
FR
WT
Pdcd1
–/–
Divided (%)
25
50
75
100
h i
0
100
200
300
400
500
600
α-CD3
WT T
FH
1:1 T
FR
+ + + +
+ +
2
µg/ml
10
µg/ml
IgG (ng/ml)
WT
T
FH
WT
T
FH
WT
T
FH
1:5 T
FR
Pdcd1
–/–
k
0
50
100
150
200
250
CD4 T
IgG (ng/ml)
*
WT
IgG (ng/ml)
CD4 T
WT
T
FH
WT
T
FH
WT
T
FH
1:1 T
FR
Pdcd1
–/–
0
50
100
150
800
1,000
1,200
1,400
Naive
j
*
*
*
**
NS
WT
CD4
T
FH
Foxp3
Gated on CD4
+
CD19
ICOS
+
CXCR5
+
T
FR
T
FR
a
GITR
T
FH
T
FR
GITR
CD4
Gated on CD4
+
CD19
ICOS
+
CXCR5
+
b
15.9
71.3
1
2
3
Foxp3 mRNA
Naive
T
FH
T
FR
c
2
4
6
8
10
12
14
Bcl-6 (10
2
MFI)
NS
NS
WT
Pdcd1
–/–
WT
WT
Pdcd1
–/–
Pdcd1
–/–
Naive T
FH
T
FR
Naive T
FH
T
FR
Prdm1 mRNA
2
4
6
d
WT
Pdcd1
–/–
WT
WT
Pdcd1
–/–
Pdcd1
–/–
Naive T
FH
T
FR
Rorc mRNA
25
75
125
175
*
e
WT
Pdcd1
–/–
WT
WT
Pdcd1
–/–
Pdcd1
–/–
Naive T
FH
T
FR
Irf4 mRNA
5
10
15
20
25
*
f
WT
Pdcd1
–/–
WT
WT
Pdcd1
–/–
Pdcd1
–/–
T
FR
WT
Pdcd1
–/–
1
2
3
4
5
6
g
CD69 of T
resp
(10
3
MFI)
Figure 4 PD-1-deficient T
FR
cells
have enhanced regulatory ability.
(a) GITR expression by T
FR
cells and
T
FH
cells from the draining lymph
nodes of wild-type mice 7 d after
immunization with MOG.
(b) Expression of Foxp3 mRNA
(right) in sorted T
FR
(CD4
+
GITR
+
ICOS
+
CXCR5
+
CD19
) cells and T
FH
(CD4
+
GITR
ICOS
+
CXCR5
+
CD19
)
cells (sorted at left), presented
relative to that on naive (CD4
+
ICOS
CXCR5
) cells, set as 1. (c) Bcl-6
protein on T
FH
cells and T
FR
cells
from wild-type and Pdcd1
−/−
mice.
(df) Expression of Prdm1 mRNA (d), Rorc mRNA (e) and Irf4 mRNA (f) in wild-type and Pdcd1
−/−
T
FR
and T
FH
cells, presented relative to that in
naive wild-type cells. (g) CD69 expression and (h) proliferation (assessed by CFSE dilution) of responder T cells in cultures of T
FR
cells obtained
from the draining lymph nodes of MOG-immunized wild-type and Pdcd1
−/−
mice and plated for 4 d (at a ratio of 1:1:1) with CFSE-labeled wild-type
(CD4
+
CD62L
+
Foxp3
) responder cells and GL7
B cells, plus anti-CD3 and anti-IgM. (i) IgG in supernatants of cultures of GL7
B cells obtained from
the draining lymph nodes of MOG-immunized wild-type mice, incubated with wild-type T
FH
cells in the presence (+) or absence (−) of T
FR
cells, plus
anti-IgM and various doses (bottom) of anti-CD3 (α-CD3). (
j) IgG in supernatants of cultures as in i, with or without naive (CD4
+
ICOS
CXCR5
CD19
)
T cells from MOG-immunized wild-type mice (left two bars) or with wild-type T
FH
cells in the presence or absence of wild-type or Pdcd1
−/−
T
FR
cells
(at a ratio of 1:1; right three bars), plus anti-CD3 and anti-IgM. (k) IgG in culture supernatants of GL7
B cells obtained as in i and plated for 6 d
with wild-type T
FH
cells in the presence or absence of wild-type or Pdcd1
−/−
T
FR
cells (at a ratio of 1:5), plus anti-CD3 and anti-IgM. *P < 0.05 and
**P < 0.005 (unpaired Student’s t-test). Data are representative of three experiments (a,c; error bars (c), s.e.m.), two experiments (b), (df; mean
and