Download Dendritic cells expand antigen-specific Foxp3+ CD25+ CD4+

Sayuri Yamazaki
Kayo Inaba
Kristin V. Tarbell
Ralph M. Steinman
Dendritic cells expand antigenspecific Foxp3+CD25+CD4+
regulatory T cells including
suppressors of alloreactivity
Authors’ addresses
Summary: Thymic derived naturally occurring CD25+CD4+ T regulatory
Sayuri Yamazaki1, Kayo Inaba2, Kristin V. Tarbell1,
Ralph M. Steinman1
cells (Tregs) suppress immune responses, including transplantation. Here
we discuss the capacity of dendritic cells (DCs) to expand antigen-specific
Tregs, particularly polyclonal Tregs directed to alloantigens. Initial studies
have shown that mature DCs are specialized antigen-presenting cells
(APCs) for expanding antigen-specific CD25+ CD4+ Tregs from TCR
transgenic mice. When triggered by specific antigen, these Tregs act
back on immature DCs to block the upregulation of CD80 and CD86
costimulatory molecules. More recently, DCs have been used to expand
alloantigen-specific CD25+CD4+ Tregs from the polyclonal repertoire in
the presence of interleukin-2 (IL-2). Allogeneic DCs are much more
effective than allogeneic spleen cells for expanding CD25+CD4+ Tregs.
The DC-expanded Tregs continue to express high levels of Foxp3, even
without supplemental IL-2, whereas spleen cells poorly sustain Foxp3
expression. When suppressive activity is tested, relatively small numbers
of DC-expanded CD25+CD4+ Tregs exert antigen-specific suppression in
the mixed leukocyte reaction (MLR), blocking immune responses to the
original stimulating strain 10 times more effectively than to third party
stimulating cells. DC-expanded Tregs also retard graft versus host disease
(GVHD) across full major histocompatibility complex (MHC) barriers.
In vitro and in vivo, the alloantigen-specific CD25+CD4+ Tregs are much
more effective suppressors of transplantation reactions than polyclonal
populations. We suggest that the expansion of Tregs from a polyclonal
repertoire via antigen-presenting DCs will provide a means for antigenspecific control of unwanted immune reactions.
1
Laboratory of Cellular Physiology and
Immunology, Chris Browne Center of
Immunology and Immune Disease, The
Rockefeller University, New York, NY, USA
2
Department of Animal Development and
Physiology, Graduate School of Biostudies,
Kyoto University, Kyoto, Japan
Correspondence to:
Sayuri Yamazaki
Laboratory for Cellular Physiology
Immunology
The Rockefeller University
1230 York Avenue
Box 176
New York, NY 10021
Tel.: +1 212 327 8875
Fax: +1 212 327 8106
E-mail: [email protected]
and
Keywords: dendritic cells, CD25+CD4+ regulatory T cells, tolerance, Foxp3, GVHD,
mixed leukocyte reaction
Introduction
Immunological Reviews 2006
Vol. 212: 314–329
Printed in Singapore. All rights reserved
ß 2006 The Authors
Journal compilation ß 2006 Blackwell Munksgaard
Immunological Reviews
0105-2896
314
The suppressive activity of thymic derived naturally occurring
CD25+CD4+ regulatory T cells (Tregs) was first noted in
the context of reactivity to self- and environmental antigens
(1–7). Two seminal observations were that adoptive transfer
of CD25-depleted T cells into nude mice generated organspecific autoimmune diseases such as gastritis, oophoritis, and
thyroiditis and that autoimmunity was prevented by
Yamazaki et al Dendritic cells expand antigen-specific Tregs
cotransfer of CD25+CD4+ T cells (8, 9). Thus, autoreactivity is
an intrinsic feature of peripheral T cells, but autoimmune
disease is prevented by natural Tregs. CD25+CD4+ Tregs
were then shown to regulate not only autoimmunity but
also tumor immunity, antimicrobial resistance, allergy, and
transplantation reactions (10–16).
The early research from the groups of Sakaguchi (17) and
Shevach (18, 19) showed that T-cell receptor (TCR) signaling
was required for CD25+CD4+ Tregs to exert suppression, but
once activated, the Tregs could block the response of
CD25–CD4+ T cells to other specificities. To study antigen
specificity, Takahashi et al. (17) tested suppression by
CD25+CD4+ Tregs on CD25–CD4+ T cells from two types of
ovalbumin (OVA) CD4 transgenic mice, DO11.10 mice
responding to OVA323-329 peptide, and BOG mice responding to OVA271-285 peptide. CD25+CD4+ Tregs from DO11.10
mice suppressed the response of CD25–CD4+ T cells from BOG
mice in the presence of both peptides but did not exert suppression with BOG peptide alone (17). The reciprocal experiment with CD25+CD4+ Tregs from BOG mice showed
suppression only in the presence of mixed peptides but not
with DO11.10 peptide alone. Thornton et al. (19) did similar
experiments with hemagglutinin (HA)-specific and pigeon
cytochrome C (PCC)-specific CD4+ transgenic mice and also
showed that a mix of peptides was necessary for suppressors
specific for one antigen to suppress CD25–CD4+ Tregs for
another antigen. Therefore, the antigen specificity of
CD25+CD4+ Tregs could be predicted to occur at the induction
phase of the suppression but not at the effector phase.
Nonetheless, much of the research in this field has utilized
bulk populations of CD25+CD4+ Tregs rather than antigenspecific ones. Interestingly, prior to the discovery that CD25
was a useful marker for Tregs, it was clear from the work of
North and colleagues (20) that suppressor cells for tumor
immunity were antigen specific and that these suppressors
were induced in tumor-bearing mice. If the contribution of
Tregs to the immune system is to be investigated in an
optimal way, antigen-specific Treg populations will need to
be addressed. For example, if one attempted to improve
tumor immunity by depleting all Tregs on the basis of CD25
expression, one would also be removing Tregs specific for
autoantigens. Reciprocally, if one attempted to block autoimmunity or graft versus host disease (GVHD) by expanding or
administering total CD25+CD4+ T cells, suppression of tumor
and microbial resistance might also occur. Thus, the biology
of antigen-specific Tregs will likely need to be deciphered to
understand the generation of suppression in different settings
and to harness these cells in immunotherapy.
Here, we review research on antigen-specific Tregs that
has come about as a result of recent research on the antigen-presenting cell (APC) requirements for expanding Tregs in the
periphery. We emphasize T cells that regulate alloreactivity,
particularly suppression of the mixed leukocyte reaction
(MLR) and GVHD, to illustrate the concept that dendritic
cells (DCs) are able expand functional antigen-specific Tregs
from the polyclonal repertoire. The expanded Tregs are
much more effective than polyclonal CD25+CD4+ populations
in regulating immunity and do so in an antigen-specific
manner.
A role for DCs in controlling antigen-specific Tregs
from TCR transgenic mice
OVA-specific TCR transgenic T cells
To study the role of APCs in the control of Treg function, TCR
transgenic mice were initially used. In the case of OVAspecific CD4 transgenic mice (termed DO11.10 and OT-II),
we (21) and others (22, 23) found that CD25+CD4+ Tregs
proliferated when challenged with mature DCs that were
presenting processed OVA protein. This finding was surprising because previously, a characteristic feature of CD25+CD4+
Tregs was their anergy in response to TCR stimulation, i.e.
they failed to proliferate in response to splenic APCs in vitro
(17–19). In contrast, CD86high mature bone marrow (BM)
DCs as well as CD11c+ DCs from mice injected with complete
Freund’s adjuvant, induced substantial expansion of
CD25+CD4+ Tregs in vitro (21). It was noted that mature
DCs were much more effective than immature cells,
whereas B cells and macrophages were inactive as APCs. The
antigen-dependent proliferation of CD25+CD4+ Tregs was
partially dependent on CD80/CD86 expression by DCs
and interleukin-2 (IL-2) by DCs and/or T cells (21).
Importantly, the DC-expanded Tregs were able to suppress
CD25–CD4+ OVA-specific TCR transgenic cells responding to
spleen APCs in vitro, and these cells were actually more active
on a per cell basis than the freshly isolated unexpanded
populations.
To test the specificity of DC-expanded Tregs, we performed
experiments along the lines of prior reports (17, 19). We
used BM-derived DCs from BALB/c mice to expand either
DO11.10 OVA-specific CD25+CD4+ T cells or 6.5 HA-specific
CD25+CD4+ T cells with the respective OVA and HA peptides,
as described previously (21). Each T cell could suppress
proliferation of the corresponding CD25–CD4+ T cells in the
presence of the specific peptide (data not shown). Then we
mixed Tregs of one specificity, e.g. OVA, with CD25–CD4+ T
Immunological Reviews 212/2006
315
Yamazaki et al Dendritic cells expand antigen-specific Tregs
cells of the other specificity, e.g. HA (Fig. 1). The addition of
HA peptide allowed the HA-specific CD25+CD4+ T cells to
proliferate, but the Tregs blocked the proliferation of the
Proliferation of
HA-specific
OVA-specific
CD25+ T cells
CD25– T cells
104
No Ag
% of max
103
102
101
100 0
10
104
101
102
103
104
100
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102
103
104 100
101
102
103
104
101
102
103
104
HA + OVA
% of max
103
102
101
100
100
104
101
102
103
100
104
HA + OVA
% of max
103
102
100 0
10
104
101
102
103
104
100
101
102
103
104
HA + OVA
103
% of max
CD25– T (PKH)
101
102
101
100
100
101
102
103
104
BDC2.5 transgenic T cells specific for an islet b cell
autoantigen
100
101
102
103
104 100
101
102
103
104
CD25+ T (CFSE)
Fig. 1. Tregs specific for one antigen suppress CD25–CD4+ T cells
with a different specificity. CD25+CD4+ T cells from 6.5 hemagglutinin (HA)-specific CD4+ transgenic mice were cultured with
BALB/c BM-dendritic cells (DCs) and HA peptide(3 mg/ml) plus
100 U/ml IL-2, as described (21). After 1–2 weeks expansion, the
DCs were depleted with anti-CD11c MACS microbeads (Miltenyi
Biotech, Bergisch Gladbach, Germany) to provide >98% pure DCexpanded Tregs. These were labeled with carboxyfluorescein diacetate
succinamidyl ester (CFSE) and added to an equal number (104) of
PKH26 labeled CD25–CD4+ T cells from DO11.10 ovalbumin (OVA)
CD4 transgenic mice. The two types of T cells were cultured with 104
CD11c+ spleen DCs in the presence of OVA protein (100 mg/ml) and
HA peptide (3 mg/mL) for 4 days, before FACS analysis. The T cells
did not divide without any antigen (top). DC-expanded, HA-specific,
CD25+CD4+ T cells proliferated in response to HA (second row),
while OVA-specific, CD25–CD4+ T cells from responded to OVA
(third row, middle). The mixture of OVA-specific CD25–CD4+ T cells
and DC-expanded, HA-specific CD25+CD4+ Tregs suppressed
CD25–CD4+ T-cell proliferation (arrow, bottom). The plots on the left
are gated on live CD4+ T cells, the middle histograms are gated on
PKH26+ DO11.10 CD25–CD4+ T cells, and the right histograms are
gated on CFSE+ DC-expanded, HA-specific, CD25+CD4+ Tregs. Similar
results were also obtained with OVA peptide (0.001–0.005 mg/ml).
Shown is one result of three similar experiments.
316
OVA-specific CD25–CD4+ T cells to OVA protein. In other
words, once the TCR of Tregs is engaged by its antigen, the
Treg is able to suppress responses of CD25–CD4+ T cells
specific for another antigen (Fig. 1).
A puzzling point with TCR transgenic T cells was the significant expansion of CD25+CD4+ Tregs in the absence of
exogenous IL-2, although IL-2 boosted proliferation significantly (21). While Tregs produce little or no IL-2, small
amounts of IL-2 were found in the cocultures of DCs and
Tregs. One possibility was that the DCs were a source of IL-2,
but DCs from IL-2–/– mice remained active. Another possibility was that all of the Tregs could produce small amounts of
IL-2, which is known to be essential for maintenance of Tregs
in vivo (24–27). A third possibility, which we favor, is that
selection of Tregs on the basis of CD25 expression failed to
provide totally pure populations, since up to 10% of the
selected CD25+ cells do not express Foxp3. The production
of IL-2 from Foxp3– cells may have driven the expansion of
the Foxp3+ Tregs (24). In contrast to the original studies with
OVA-specific TCR transgenic T cells, the need for exogenous
IL-2 in DC-based expansion of antigen-specific Tregs is quite
striking when one turns to polyclonal populations, as will be
described for the MLR below.
Immunological Reviews 212/2006
Mature DCs were then used to expand antigen-specific
CD25+CD4+ Tregs from BDC2.5 transgenic mice, which are
reactive to an antigen (as yet unidentified) within pancreatic
islet b cells. This system allows one to study the capacity of the
expanded Tregs to suppress spontaneous autoimmunity, i.e.
insulin-dependent diabetes mellitus, in non-obese diabetic
(NOD) mice. Again, mature DCs expanded Treg numbers
5–10-fold over a week in culture in the presence of IL-2 and
a peptide mimetope for the naturally processed b cell antigen.
The Tregs prevented the development of diabetes induced by
polyclonal diabetogenic T cells from NOD mice (28). The
antigen-specific, DC-expanded, CD25+CD4+ Tregs were at
least 100 times more active in preventing diabetes than polyclonal CD25+CD4+ Tregs from NOD mice, even if the latter
were activated with DCs and anti-CD3. These findings of
Tarbell et al. indicate that antigen-specific Tregs are much
more effective than bulk populations of CD25+CD4+ T cells
in regulating the immune system in vivo, including the
regulation of autoimmune disease. The function of antigenspecific Tregs in controlling autoimmunity is reviewed
elsewhere (29, 30).
Yamazaki et al Dendritic cells expand antigen-specific Tregs
CD25+
DC alone
Peptide
CD25–
CD25+
& CD25–
+
–
CD86
Class II
+
–
CD80
Class II
+
104
103
control
Although antigen-specific Tregs are likely to be critical in
regulating immune responses to antigens in self-tissues,
tumors, transplants, and infections, the literature clearly indicates that once the Treg is triggered, suppression extends to
immune responses elicited by other antigens presented on the
same APC (17). Misra et al. (31) addressed the underlying
mechanism by activating CD25+CD4+ Tregs from human
blood with anti-CD3 and then showing that these Tregs
could suppress several components of DC maturation that
take place when DCs are responding to CD40 ligation. The
features of maturation that were inhibited by the Tregs
included the upregulation of CD80, CD83, and CD86 [but
the increase in major histocompatibility complex (MHC) class
II and CD40 was not blocked]. Similarly, freshly isolated
mouse CD25+CD4+ Tregs downregulated CD80 and CD86
on DCs and B cells (32). To examine the effects of Tregs on
DCs that were presenting a cognate antigen, we expanded TCR
transgenic OVA-specific Tregs and added these to freshly
isolated spleen DCs that were presenting OVA to activated
CD25–CD4+ T cells. Spleen DCs mature in culture, but the
upregulation of CD80 and CD86 was clearly blocked by the
addition of Tregs in the presence of peptide (Fig. 2). This
finding suggests that Tregs are able to dampen the costimulatory function of DCs, which would allow Tregs triggered by
one antigen to block the effective presentation of other antigens by the same APCs.
Recent experiments have begun to look at the interaction of
Tregs with DCs in vivo. CD25+CD4+ Tregs, capable of suppressing inflammatory bowel disease, were noted to interact with
CD11c+ DCs in sections of the mesenteric lymph nodes and
colon (33). Two-photon microscopy has now been used to
follow cellular interactions in intact lymph nodes. BDC2.5
transgenic Tregs, which are reactive with an islet b cell autoantigen, interact with antigen-presenting DCs in explanted
pancreatic lymph nodes, and this interaction leads to reduced
clustering of antigen-specific autoreactive CD25–CD4+ T cells
with antigen-bearing DCs (34). Similarly, in myelin basic
protein (MBP) transgenic mice, the injection of MBP-specific
Tregs reduced the stable contacts that normally develop
between antigen-specific CD4+ T cells and antigen-loaded
injected DCs in fully intact lymphoid tissues (35). These
reports indicated that Tregs interact with DCs in vivo and
suppress presentation to effector T cells.
Fallarino et al. (36) have provided one potential mechanism
of immune suppression via DCs that interact with Tregs,
A
–
102
101
100
100
101
102
103
104
Class II
Gated on CD11c+ cells
B
100
% of max
Antigen-specific Tregs block the increased expression of
CD80 and CD86 costimulatory molecules on maturing DCs
in vitro and DC-effector T-cell binding in vivo
CD25–
80
CD25–
& CD25+
60
40
20
0
100
101
102
103
104
CD86
Gated on
CD80
control
CD11c+ DCs
Fig. 2. Dendritic cell (DC) maturation is suppressed by culture with
DC-expanded Tregs in vitro. (A) As in Fig. 1, CD11c+ DCs were selected
from spleen by CD11c-MACS and cultured with DC-expanded CD25+ or/
and CD25–CD4+ T cells from ovalbumin (OVA) DO11.10 CD4+ transgenic mice in the presence (+) or absence (–) of OVA peptide. After 2
days, the cultures were stained for CD11c, MHC class II, CD86, CD80, or
isotype control antibody. In the presence of CD25–CD4+ T cells and
peptide, a subset of DCs (arrows) matures, and this maturation is blocked
by addition of Tregs (right vertical row). Plots are shown for gated
CD11c– cells. Shown is one of three similar experiments. (B) As in (A),
expression of CD86 or CD80 was compared in culture with peptide.
Histograms are shown gated on CD11c+ DCs and the arrows denote the
maturing DCs without the addition of Tregs.
involving indoleamine 2,3-dioxygenase. This enzyme catabolizes tryptophan, leading to decreased tryptophan levels as
well as toxic kyneurenine metabolites, both of which may
Immunological Reviews 212/2006
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318
Immunological Reviews 212/2006
RAG–/–
Aly/aly–/– + splenectomy
Lethally irradiated
Lethally irradiated
Lethally irradiated
Sublethally irradiated
Skin graft
Skin graft
GVHD with BMT 2x106
GVHD with BMT 5 106
GVHD with BMT 5 106
GVHD without BMT
Summary of literature using naı̈ve polyclonal CD25+CD4+ Tregs to suppress transplant reactions in vivo. The experiments have been done mainly with skin graft and graft versus host disease (GVHD)
with or without bone marrow transplantation (BMT). The table summarizes the type of recipients as well as the numbers of injected effector T cells and CD25+CD4+ Tregs.
Jarvinen et al. (81)
Dai et al. (105)
Hoffmann et al. (13)
Cohen et al. (71)
Edinger et al. (106)
Taylor et al. (14)
Yes
Yes
Yes
No
No
Yes
No
No
Yes
Yes
Yes
Weak
3-5:1
3:1
2:1
5:1
1:1
4:1
1:1
1:1
1:1
0.5:1
1:1
1:1
1 106
6 105
1 105
5 105
4 105
4 105
2 105
1 106
4.5 105
5 106
5 105
1 105
Spleen, 1 107
CD4+ T, 2 105
CD4+ T, 5 104
CD45RBhigh CD4+ T, 1 105
CD25– CD4+ T, 4 105
1 105
CD25– CD4+ T, 2 105
Donor specific memory CD8, 1 106
CD25– CD4+T, 4.5 105
T, 10 106
T, 5 105
CD25– CD4+ T, 1 105
T-depleted
Nude
RAG–/–
T-depleted
SCID
graft
graft
graft
graft
graft
Skin
Skin
Skin
Skin
Skin
Number of naı̈ve Treg
Recipient mice
Systems
Transplantation immunity provides an important setting for
the study of Treg function, especially the importance of antigen-specific Tregs within polyclonal rather than TCR transgenic populations. The reason is twofold. First, the frequency
of alloreactive T cells is high in contrast to nominal antigens,
presumably because the term ‘alloantigen’ likely comprises
hundreds even thousands of allo-MHC peptide complexes
that cross-react with self-MHC-peptide. Second, transplantation provides a chance to study suppression of cell-mediated
immunity in vivo, either in host versus graft or graft versus host
reactions.
In Table 1, we list publications in which polyclonal
CD25+CD4+ Tregs from naı̈ve mice have been tested for
suppression of GVHD and rejection of allogeneic skin grafts.
The experimental models all involved irradiated or lymphopenic recipients of effector T cells, which would induce
GVHD or reject skin. The use of these so-called empty hosts
could provide a driving force for the expansion and activation
of the transferred CD25+CD4+ Tregs that are used to suppress
alloreactivity. The underlying phenomenon is termed
‘homeostatic expansion’, wherein cytokines like IL-7 help
reestablish normal lymphocyte numbers in lymphopenic
hosts. While Tregs have been reported to express lower levels
of IL-7 receptors and exhibit poor homeostatic proliferation
(37), it is also possible that the TCRs of polyclonal alloreactive
CD25+CD4+ Tregs are able to cross-react with recipient MHC
molecules to stimulate expansion in lymphopenic mice.
Nonetheless, the experimental design in the references in
Table 1 does not allow one to assess the relative importance
of antigen-specific Tregs in the suppression of transplantation
immunity, because the selection for antigen-specific Tregs
may have occurred in the recipients following adoptive
transfer.
Table 1. Suppression of transplantation reactions in vivo by polyclonal CD25+CD4+Tregs
Polyclonal CD25+CD4+ Tregs suppress transplantation
immunity
Type and number of effector T cells
Ratio Treg: effect
Suppression
References
suppress T-cell function. They found that cytotoxic Tlymphocyte antigen-4 (CTLA-4) on the Tregs activates
tryptophan catabolism via CD80/86 on DCs (36).
Together, these studies are shifting the mechanism of action
of Tregs from a direct dampening of other effector T cells to a
mechanism that involves DCs. The latter is more attractive to
us, since DCs are able to present antigen in a specific way on
MHC class II to CD4+ T cells, whereas at least in mice, effector
T cells do not express antigen-presenting class II products and
would seem unable to specifically select antigen-specific
Tregs.
Graca et al. (103)
Nishimura et al. (73)
Graca et al. (104)
Kingsley et al. (78)
Sanchez-Fueyo et al. (80)
Yamazaki et al Dendritic cells expand antigen-specific Tregs
Joffre et al. (38)
Yamazaki et al. (39)
Cohen et al. (71)
Trenado et al. (72)
Yes
Yes
2–3:1
1:1
Yes
1:1
Spleen, 5 104
DC, 1–2 105
Spleen, 1 105
CD25– CD4+
T cells, 1–2 105
T, 10 10
Lethally irradiated
Lethally irradiated
Sublethally irradiated
Spleen, 10 10
6
BMT
GVHD without BMT
GVHD with BMT 5 10
0.7:1
T, 10 106
6
6
Yes
Antigen specific
at 1:32–64
Antigen non-specific
at 1:4
Antigen non-specific
at 1:1
NT
Antigen specific
at 1:32–64
Yes
5–1:1
Spleen, 106 or 2 105,
7d before effectors
Spleen, 7 106
T, 2 105
Suppression in vitro
Antigen-specific
suppression in vivo
Ratio Treg to
effector T cells
Number of expanded Treg
Type and number
of effector T cells
Lethally irradiated
We purified CD25+CD4+ Tregs from spleens and lymph
nodes and stimulated them with mature allogeneic BM-DCs
in the MLR. We found a strong proliferative response when
GVHD with BMT 5 106
alloreactive
Nude
of
Skin graft
DCs are more potent stimulators
CD25+CD4+ Tregs than spleen APCs
Recipient mice
We recently have found that alloantigen-specific
Foxp3+CD25+CD4+ Tregs from a polyclonal repertoire can
be expanded by DCs much better than with splenic APCs,
which only contain 1–2% DCs (39). In the MLR, DCs could
expand the total number of alloreactive CD25+CD4+ Tregs
three to fivefold in a week, while splenic APCs did not expand
cell numbers at all. The DC-expanded CD25+CD4+ Tregs, in
contrast to unexpanded cells, could suppress both the MLR in
vitro and GVHD in vivo in an antigen-specific manner.
System
Efficient expansion of alloantigen-specific CD25+CD4+
Tregs by DCs
Table 2. Suppression of transplant reactions with CD25+CD4+ Tregs selected by allogeneic spleen or DCs
Several groups have used allogeneic APCs plus IL-2 to expand
antigen-specific CD25+CD4+ Tregs before testing their suppressive function in vivo (Table 2). The goal is to use allogeneic
spleen cells to increase the frequency of antigen-specific
Tregs. To achieve effective suppression, a wide range of
Treg
numbers
have
been
used
CD25+CD4+
4
6
(5 10 10 10 ), generally with comparable numbers
of effector T cells (Table 2). In some of the in vivo experiments,
third party APC-expanded Tregs were tested to assess antigenspecific suppression. Although splenic APC-expanded Tregs
seemed to show antigen-specific suppression in vivo, it is of
note that relatively large numbers of expanded Tregs were
used, which is in contrast to the number of fresh polyclonal
Tregs used in the studies in Table 1. The one experiment in
which small numbers of spleen expanded Treg (5 104)
seemed to show antigen-specific suppression in vivo was by
Joffre et al. (38). They transplanted three sources of BM cells
(H-2b B6, H-2b H-2d B6 D2 F1, and H-2b H-2k
B6 CBA F1) into irradiated H-2b B6 hosts with effector
H-2b B6 spleen cells. The mice that received B6 CD25+CD4+
Tregs expanded by B6D2 F1 splenic APCs suppressed rejection
of the H-2b H-2d B6D2 F1 BM but not rejection of the H2b H-2k B6CBAF1 BM (38). The reciprocal experiments
also showed antigen-specific suppression, leading to the conclusion that antigen-specific CD25+CD4+ Tregs can suppress
rejection in an antigen-specific manner in vivo (38).
References
Function of CD25+CD4+ Tregs that are first expanded
by allogeneic spleen cells
Nishimura et al. (73)
Yamazaki et al Dendritic cells expand antigen-specific Tregs
Immunological Reviews 212/2006
319
Yamazaki et al Dendritic cells expand antigen-specific Tregs
IL-2 was added to the MLR cultures, but in contrast to DCs,
the proliferation was weak if allogeneic spleen cells were used
as APCs (Fig. 3A). CD25+CD4+ Tregs also responded to syngeneic DCs in the presence of exogenous IL-2. The proliferation of Tregs induced by mature DCs was dependent on the
dose of added IL-2 (Figs 3A and B) (39). When the recovery of
live T cells was followed (the DCs from the cocultures were
removed using anti-CD11c coated magnetic beads), there was
about a three to fivefold expansion relative to the initial input
of Tregs in 1 week (Fig. 3C). If the recovered Tregs were
restimulated with fresh mature DCs plus 500 U/ml IL-2, the
T cells continued to expand in numbers, with a 20–25 fold
expansion by 3 weeks (Fig. 3C). When we scaled up the
cultures from 96-well to 24-well plates, using the same initial
concentration of Tregs (5 104/ml) and IL-2 (500 U/ml), a
similar expansion in cell numbers was observed in response to
mature DCs (data not shown).
It is important to distinguish two components of DC function in these cultures; where the Tregs are expanding in total
numbers and also on a per cell basis, the Tregs are being
enriched some 10-fold in antigen-specific suppressive activity.
One component is that the DCs are presenting antigen, providing a selection pressure for preferential expansion of antigen-specific cells. The other is that DCs also can expand Tregs
in the presence of IL-2 but in the ostensible absence of antigen. For example, OVA-specific TCR transgenic Tregs (but not
CD25– TCR transgenic T cells) are able to expand when DCs
are added without OVA antigen (21), and polyclonal Tregs
expand to syngeneic DCs (21, 39). Therefore, DCs are exerting both antigen-specific and non-specific effects on Tregs in
vitro.
CD25+ CD4+
A
20 Day 7
10 Day 3
8
No IL-2
IL-2 100 U/ml
IL-2 500U/ml
3H-TdR
6
uptake
(cpm
4
×104)
12
8
2
4
0
B
16
T
Sp
Sp BMalone APC CD11c+ DC
DC
0
T
Sp
Sp BMalone APC CD11c+ DC
DC
CD86+ BM-DCs
16
IL-2 500 U/ml
IL-2 100 U/ml
12
IL-2 20 U/ml
No IL-2
Fold
increase 8
4
0
C
3
5
7
Days after culture
BALB CD25+ CD4+ stimulated with IL-2 500 U/ml
20
16
1st and 2nd 1:1
B6 CD86+ DC
12
1st and 2nd 1:1
BALB CD86+ DC
Fold
expansion
Restimulation
8
4
+
+
Phenotype of CD25 CD4 Tregs after expansion with allogeneic DCs plus IL-2
+
+
CD25 CD4 Tregs constitutively express CTLA-4 (40–42) and
GITR [glucocorticoid-induced tumor necrosis factor (TNF)
receptor family related gene] (43, 44). A proportion of the
CD25+CD4+ T cells also express the CD62L lymph node
homing selectin (45–48) and the CD103 aEb7-integrin (49).
Integrin aEb7–CD25+ CD4+ Tregs express CD62L and are able
to migrate to lymph nodes efficiently, whereas integrin aEb7+
Tregs with low CD62L express high E/P-selectin-binding
ligands and enter into inflammatory sites (50). CD103+
Tregs also play an important role in suppressing effector
cells specific for Leishmania major (51). Recently, it was shown
that CD103 must be expressed on non-Tregs, specifically on
intestinal DCs, during suppression of colitis induced by
CD45RBhighCD4+ T cells (52).
320
Immunological Reviews 212/2006
0
0
5
10
15
20
Days after culture
25
Fig. 3. Allogeneic dendritic cells (DCs) but not spleen antigen-presenting cells (APCs) effectively expand CD25+CD4+ T cells together
with IL-2. (A) 104 BALB/c CD25+CD4+ T cells were cultured with 104 B6
BM-DCs, 104 spleen CD11c+ DCs, or 105 splenic APCs. The DCs were
added to T cells in the absence (open bars) or presence (shaded and black
bars) of the indicated doses of rhIL-2 for 3–7 days. 3H-thymidine uptake
was assessed for the last 12 h (39). (B) As in (A), BALB/c CD25+CD4+ T
cells were cultured with B6 BM-DCs in the presence of the indicated doses
of IL-2. T-cell proliferation was measured as live cell counts per culture in
triplicate wells on each day. Fold increases compared to the T-cell number
on day 0 were calculated. (C) 104 BALB/c CD25+CD4+ T cells that had
been cultured with 104 B6 or BALB BM-DCs plus IL-2 for day 7 were
washed twice, and DCs were eliminated with CD11c beads. The recovered
live T cells were cultured with fresh DCs plus IL-2 for another 14 days
(104 T cells, 104 DCs, in 96 round wells). Fold increases compared to the
T-cell number on day 0 was calculated after MACS depletion of CD11c+
DCs on day 7 and day 21. Shown is one result of two similar experiments.
Yamazaki et al Dendritic cells expand antigen-specific Tregs
DCs maintain Foxp3 expression by expanded CD25+CD4+
Tregs
Several reports indicate that the homing of Tregs is a key
component for antigen-specific suppression in vivo (53).
CD62L–CD25+CD4+ Tregs are weak suppressors in vivo for
GVHD and type I diabetes (46–48). CD62L allows for
lymph node homing and interaction with antigen-bearing
DCs. Lymph nodes are required to induce tolerance, and
anti-CD62L antibody can block the induction of tolerance
(54). We therefore purified the CD62L+ and CD62L– fractions
of CD25+ CD4+ T cells and then expanded these with IL-2 and
allogeneic DCs. As mentioned below, the CD62L– fractions of
mouse spleen CD25+CD4+ T cells have a significant contamination with Foxp3– cells. Strong proliferation of both
CD62L+ and CD62L– fractions was induced by DCs (39).
After 1 week of culture, the typical markers of Tregs were
retained following expansion with either allogeneic or syngeneic DCs, i.e. the T cells maintained high CD25, CD62L,
and GITR levels (Fig. 4A). Expression of CTLA-4 was also
maintained (data not shown). When the DCs were used to
expand CD62L–CD25+CD4+ T cells, some cells expressed
CD62L and a few downregulated CD25 (Fig. 4A). A few cells
in the CD62L– subset but not the CD62L+ subset expressed the
CD103 integrin (39). Interestingly, when DCs were cultured
with Tregs for a week, the Tregs upregulated the mucosal
homing integrin a4b7 (unpublished data and Fig. 4B).
However, there is evidence that expression of b7 integrin on
CD25+CD4+ Tregs is not required to suppress colitis in an
inflammatory bowel disease system, although b7 integrin is
necessary
for
the
function
of
disease-inducing
high
+
CD45RB CD4 T cells (55). Coupled with the data on
Foxp3 expression below, it is evident that natural Tregs retain
their expected phenotype upon expansion with DCs.
Coculture with DCs helped CD25+CD4+ Tregs maintain and
even increase Foxp3 expression, whereas spleen APCs were
much less effective (39). Foxp3 is a key transcription factor
that controls the development and function of naturally occurring CD25+CD4+ Tregs (56–60). Monoclonal anti-mouse
Foxp3 antibodies have recently become available, and intracellular fluorescence-assisted cell sorter (FACS) staining has
shown expression of Foxp3 on >90% of mouse spleen
CD25+CD4+ T cells as well as a small fraction of
CD25–CD4+ T cells. Foxp3+CD25+ and Foxp3+CD25– cells
represented 9.8 0.7% and 2.3 0.01% of the CD4+ T
cells, respectively (n ¼ 3), which was comparable with
reports using Foxp3-green fluorescence protein knockin
mice (59, 60). CD25highCD4+ T cells were approximately
90% Foxp3+, whereas CD25intermediateCD4+ T cells were
approximately 60% Foxp3+ (Fig. 5A). After CD25high CD4+ T
cells from either spleen or mesenteric lymph node were
cultured with DCs plus with IL-2, high Foxp3 expression
was maintained, and again there was little difference between
CD25+CD4+ Tregs expanded with allogeneic or syngeneic
DCs (Fig. 5B). Interestingly, DCs maintained Foxp3 expression
in the absence of IL-2, whereas spleen APCs did not sustain
Foxp3 expression even in the presence of IL-2 (39). Foxp3
expression was maintained and even increased (relative to the
starting population of Tregs from spleen) by DCs through
CD80/CD86-independent mechanisms, because DCs from
CD80/CD86 double knockout mice were able to sustain
Foxp3 as well as DCs from control wildtype mice (Fig. 5C).
These results indicate that DCs may sustain Foxp3 in the
11
CD103
101
99.7
GITR
102
97
CD26L
CD25
99.7
103
control
104
A
0.1
100
100 101 102 103 104
CD4
B
B6 CD62L+ CD25+ CD4+
T cells from MLN
cultured with
B6 CD62L+ CD25+ CD4+ T cells
from spleen cultured with
BALB-DC + IL2
B6-DC + IL2
CBA-DC + IL2
BALB-DC + IL2
Frequency
100
80
60
40
20
0 0
10
61
101
38
102
103
α4β7 Integrin
104
79
21
55
45
62
38
Fig. 4. Phenotype of allogeneic dendritic cell
(DC)-expanded CD25+CD4+ T cells. (A) 104
BALB/c CD25+CD4+ T cells were cultured with
104 B6 allogeneic bone marrow-DCs in
500 U/ml IL-2 for 1 week. The cultures were
then stained with anti-CD4, anti-CD11c, and
the indicated antibodies. Histograms are shown
for gated, CD4+, and CD11c– cells. Shown is
one of two similar experiments. (B). As in (A),
increased a4b7 integrin expression on splenic
(left) and mesenteric lymph node (right panel)
on B6 CD62L+CD25+CD4+ T cells cultured
with the indicated DCs (freshly isolated T cells
do not express this integrin).
Immunological Reviews 212/2006
321
Yamazaki et al Dendritic cells expand antigen-specific Tregs
B6 CD62L+ CD25+
CD4+ T cells from
MLN cultured with
B
A
CD62L+
B6
8.9
CD25+
CD4+
T cells
from spleen cultured with
BALB-DC + IL2 B6-DC + IL2 CBA-DC + IL2 BALB-DC +IL2
91
94
96
93
98
0.12
0.16
0.15
0.27
Foxp3
105
CD25
104
%
of
max
3
10
2
10
104
39
103
61
Isotype10
control
2
101 102 103 104
101
100
CD4
100
100 101
80
102
103
104
CD25
60
40
98
20
2.0
0
101 102 103 104 105
Foxp3
BALB/c CD25+ CD4+ T cells cultured with
C
–IL-2
WT DC CD80/CD86 KO DC
88
83
Fresh
+IL-2
BALB/c
WT DC CD80/CD86 KO DC spleen CD4
99
99
12
Foxp3
104
Isotype
control
1.6
1.7
1.9
1.9
0.17
103
102
101
100 0
10
101
102
103
104
CD25
B6
BALB/c
19
3
10
102
1000
8.1
1000
2000
3000
CD4
Number of cells
200
70
CD62L–
100
0.73
%
of
100
max 80
99
60
0
0
102
103
104
105
CD62L
Gated on
40
20
CD25+
10
CD4+
3000
4000
CD4
300
16
200
72
100
CD62L–
0
102
103
104
105
CD62L
0 102 103 104 105
Gated on CD25+ CD4+
Foxp3
+
CD4
CD62L+
0
Fig. 5. Foxp3 expression on fresh and expanded CD25 CD4 T
cells. (A) Expression of Foxp3 on CD25 high, intermediate, and low
CD25+CD4+ T cells from mouse spleen. (B) 104 CD62L+
CD25+CD4+ splenic T cells from C57Bl/6 mice were cultured with
104 dendritic cells (DCs) from BALB/c, B6, or CBA mice plus
500 U/ml IL-2 for 1 week and stained with anti-Foxp3 antibody.
The right panel shows B6 mesenteric lymph node Tregs similarly
Immunological Reviews 212/2006
0 102 103 104 105
400
92
+
322
2000
500
0
8.6
7.4
0
1000
FSC
CD62L+
300
22
3
102
0
CD4
400
22
104
2000
0
0 102 103 104 105
4000
FSC
Number of cells
0
3000
1000
0
0
105
93
open
SSC
2000
CD25
104
open
3000
4000
105
95
SSC
4000
CD25
D
0.76
%
of 100
max 80
99
60
40
20
0
21
79
0 102 103 104 105
Foxp3
cultured with BALB/c DCs for 1 week with IL-2. (C) 104 BALB/c
CD25+CD4+ T cells were cultured with DCs from B6 wildtype
control or CD80/CD86 double knockout mice in the absence (left)
or presence (right) of exogenous IL-2500 U/ml for 7 days. Plots
were gated on CD4+ cells. Shown is one result of three similar
experiments. (D) Expression of Foxp3 in CD62L+ and CD62L–
CD25+CD4+ T cells from B6 (left) and BALB/c (right) spleen.
Yamazaki et al Dendritic cells expand antigen-specific Tregs
periphery without CD80/CD86, in contrast to a report showing that Foxp3 expression on CD25+CD4+ thymocytes
requires CD28 signaling independently of IL-2 (61). The
ability of DCs to sustain Foxp3 expression is most efficient
with CD62L+ Tregs. CD62L– Tregs are contaminated with
approximately 10–20% Foxp3– cells (Fig. 5D), and this negative fraction increases during culture with DCs and IL-2,
which may explain their reduced efficacy as suppressors of
immunity (data not shown).
All of the above studies began with natural CD25+CD4+
Tregs that had already assumed a Foxp3-driven differentiation
program. Foxp3 can be regulated by transforming growth
factor-b1 (TGF-b) (62–68). It has been reported that TGF-b
produced by DCs from tumor-bearing hosts increases the
frequency of CD25+CD4+ Tregs, which then contribute to
immune suppression (69). Recent experiments from von
Boehmer’s laboratory (70) indicate that DCs, when targeted
to present antigen in vivo via the DEC205 receptor in the
presence of TGF-b, are able to differentiate Foxp3+ Tregs
from Foxp3–CD25–CD4+ precursors. Therefore, TGF-b produced by DCs or other sources may allow DCs to differentiate
Foxp3+ Tregs in the periphery. This pathway would provide a
major route for expansion of antigen-specific Tregs.
Antigen-specific MLR suppression with CD25+CD4+
Tregs expanded by allogeneic DCs
Previous reports (71, 72) were not able to show antigenspecific suppression of the MLR in vitro by Tregs expanded
by splenic APCs, except for one study (73) (Table 2). To detect
antigen-specific suppression in vitro by DC-expanded
CD25+CD4+ Tregs, we followed the proliferation of responder CD25–CD4+ T cells by marking them for either CD45.1 or
Thy-1.2 polymorphisms and by carboxyfluorescein diacetate
succinamidyl ester (CFSE) labeling. We used this approach
because CD25+CD4+ Tregs in the cultures would be able to
take up 3H-TdR in response to the IL-2 produced by the
responder T cells in the MLR (74, 75). The suppression of
the alloMLR was then monitored by the dilution of CFSE, and
the proliferating cells were easy to distinguish from non-CFSE
labeled cells by the CD45 or Thy-1 marker. Here we show
some additional results to those published elsewhere (39).
We first cultured CD62L+CD25+CD4+ T cells from H-2b
CD45.2 B6 mice with two kinds of allogeneic DCs (H-2d
BALB/c and H-2 s SJL) plus IL-2 for 1–2 weeks (39). Then,
these expanded Tregs or freshly isolated CD62L+CD25+CD4+
T cells from B6 mice were added to CFSE-labeled responder
CD25–CD4+ T cells from CD45.1+ B6 mice, to test whether
the Tregs would suppress a primary MLR to irradiated allogeneic splenic APCs (Fig. 6A). Tregs expanded with allogeneic
DCs suppressed the alloantigen-specific MLR at lower suppressor to responder ratios relative to suppression of third party
APCs (Fig. 6A). Thus, if Tregs were expanded with SJL DCs,
they suppressed the MLR to SJL spleen APCs even at 1:27
ratios (Fig. 6A, middle). In contrast, if the Tregs were
expanded with BALB/c DCs, the MLR to SJL spleen APCs
was only suppressed at high doses, e.g. one suppressor per
three responders (Fig. 6A, right). When freshly isolated
CD62L+CD25+CD4+ T cells were compared with Tregs
expanded with allogeneic DCs, suppression was seen at high
suppressor to responder ratios (Fig. 6A, left). Likewise, alloantigen-specific suppression in the MLR to BALB/c spleen APCs
was seen with BALB/c DC-expanded Tregs, not with SJL DCexpanded Tregs (39). Taken together, these observations indicate that DCs can expand the numbers of CD25+CD4+ Tregs in
the MLR, and on a per cell basis, the expanded Tregs are much
more effective at suppressing the MLR to the same strain as the
original alloantigen-presenting DCs.
We then purified CD62L+ or CD62L–CD25+CD4+ Tregs
and expanded them with DCs plus IL-2 to compare their
suppressive activity in vitro. When CD62L–CD25+CD4+ T cells
were cultured with allogeneic or syngeneic DCs plus IL-2, the
expanded CD62L–CD25+CD4+ T cells showed less suppressive
activity for the allogeneic MLR than expanded
CD62L+CD25+CD4+ T cells (Fig. 6B), possibly because the
CD62L– fraction contains higher numbers of Foxp3– cells.
Syngeneic DC-expanded Tregs were also tested for their suppressive capacity in vitro, but suppression only occurred at a
high suppressor to responder ratio (Fig. 6B). It is of interest to
recall that Tregs expanded with syngeneic DCs have the same
phenotype, including high Foxp3 expression (39) (Fig. 5B),
to Tregs expanded with allogeneic DCs, so that antigen specificity is clearly a distinct and required component of alloDCexpanded Tregs. Our results indicate that allogeneic DCs
expand the numbers and alloantigen-specific suppressive
function of CD25+CD4+ Tregs.
Antigen-specific GVHD suppression with CD25+CD4+
Tregs expanded by allogeneic DCs
To test the suppressive function of DC-expanded Tregs in vivo,
we first used bm12 recipients and B6 donor T cells to induce
GVHD. These mice differ at three amino acids in the peptidebinding regions of the MHC class II H-2 molecule (14, 76).
Freshly isolated CD25–CD4+ T cells from B6 mice (105) were
injected into sublethally irradiated (500 rads) bm12 hosts
Immunological Reviews 212/2006
323
Yamazaki et al Dendritic cells expand antigen-specific Tregs
104
A
Responder: CD45.1 B6 CD25–
Stimulator: SJL, spleen
67
103
102
101
CD45.1
100
100
101
102
103
104
CFSE
Suppressor :
responder
66.7
SJL DC
expanded Treg
23.3
BALB DC
expanded Treg
45.4
70
27.7
62.8
72.7
44.8
69.3
Fresh Treg
1:3
1:9
with or without 104 or 105 CD25+CD4+ T cells from B6 mice
that had been expanded with bm12 DCs (Fig. 7A). All recipients injected with effector CD25–CD4+ T cells alone died by
day 15 (Fig. 7A). Mice receiving 104 bm12-DC-expanded
Tregs showed a slight prolongation of survival, with all mice
dying by day 17 (Fig. 7A). In contrast, mice receiving 105
bm12-DC-expanded Tregs survived longer. Death from GVHD
began on day 21, and all mice died by day 28 (Fig. 7A). In
another experiment, two of five recipients of 105 bm12-DCexpanded Tregs survived until day 50 (39).
We next induced GVHD in a fully MHC-mismatched BALB
and B6 combination (Fig. 7B). Sublethally irradiated BALB/c
recipients injected with effector 105 CD25–CD4+ T cells from
B6 mice alone started to die on day 14, and all mice died by
day 17. All mice receiving antigen-specifically expanded Tregs
A
104
CD25– CD4+ 1 × 105 (n = 4)
CD25– and 104 bm12-DC
exp. B6 CD25+
CD62L+ 1:0.1 (n = 4)
CD25– and 105 bm12-DC
exp. B6 CD25+
CD62L– 1:1 (n = 4)
103
1:27
102
B6
102
103
% survivial
101
104
CD25– T +
expanded Treg DC from
from
BALB B6
60 000
60
40
20
CD62–
B
40 000
0
B6
5
10
15
20
25
30
Days after GVH induction
CD25– T alone
CD25– T + BALB APC
0
1:3
1:9
CD25+: CD25–
1:27
Immunological Reviews 212/2006
40
Effector CD25– CD4–
T cells 1 × 105 (n = 4)
& Syngeneic DC exp.
Treg 3 × 105 (n = 4)
& Allogeneic DC exp.
Treg 3 × 105 (n = 4)
80
20 000
35
BALB (irradiated 500 rads)
100
Fig. 6. Allogeneic dendritic cell (DC)-expanded CD25+CD4+ regulatory T cells suppress the mixed leukocyte reaction (MLR) in vitro in an
antigen-specific manner. (A) Freshly isolated (fresh) or two kinds of
allogeneic DC (BALB-DC and SJL-DC)-expanded CD62L+CD25+CD4+ T
cells from CD45.2+ B6 mice were mixed with 105 carboxyfluorescein
diacetate succinamidyl ester (CFSE)-labeled, CD45.1+, B6, CD25–CD4+ T
cells in various ratios and stimulated with irradiated 105 spleen cells from
SJL mice. Five days later, CFSE dilution was analyzed by FACS. The
displayed cells were gated on live CD45.1+ cells and the percentage of
divided cells (in live CD45.1 cells) is shown inside the plots. Shown is
one of two similar experiments. (B) As in (A), for suppressors, CD45.2+
B6 CD62L+ or CD62L–CD25+CD4+ T cells were expanded with indicated
DCs plus IL-2. Expanded T cells were added to CFSE-labeled 105 CD45.1+
B6 CD25–CD4+ responder T cells with BALB spleen antigen-presenting
cells (APCs) as MLR stimulators. The numbers of live CD45.1+ cells/
culture are shown. Depicted is one result from two similar experiments.
324
80
0
CD62+
% survival
Number of line CD45.1 T cells/culture
100
100
B
bm12 (irradiated 550 rads)
100
101
60
40
20
0
0
5
10
15
20
25
30
35
40
45
50
Days after GVHD induction
Fig. 7. Allogeneic dendritic cell (DC)-expanded CD25+CD4+ regulatory T cells suppress graft versus host disease (GVHD) in vivo in an
antigen-specific manner. (A) 105 naı̈ve B6 CD25–CD4+ T cells were
adoptively transferred into irradiated (550 rads) bm12 recipients with or
without 104 or 105 bm12-DCs (alloantigen specific)-expanded
CD62L+CD25+CD4+ T cells. The percentages of surviving mice are
shown (n ¼ 4 in each group). Depicted is one result from two similar
experiments. (B) 1 105 naı̈ve B6 CD25–CD4+ T cells were adoptively
transferred into 500 rads irradiated BALB/c recipients with or without
3 105 BALB-DCs (alloantigen-specific) or 3 105 B6-DCs (syngeneic, antigen non-specific) expanded CD62L+CD25+CD4+ T cells.
Percentages of surviving mice are shown (n ¼ 4 in each group). Shown
is one result from two similar experiments.
Yamazaki et al Dendritic cells expand antigen-specific Tregs
(3 105 BALB-DC expanded Treg from B6) survived until
day 27; two of eight mice gained weight and survived until
day 50 (Fig. 7B). When we tested syngeneic DC-expanded
Tregs, recipients showed little or no prolongation of life
(39) (Fig. 7B). Furthermore, when we compared B6 Tregs
that had been expanded in vitro with either BALB/c H-2d DCs
or CBA H-2 k DCs, Tregs expanded with BALB DCs were more
effective than Tregs expanded with CBA DCs. Therefore, allogeneic DC-expanded Tregs show specific suppression of
GVHD in vivo, whereas third party and syngeneic DC-expanded
Tregs exhibit weaker suppression, indicating that an antigenspecific component of Treg function can be detected and
enhanced in polyclonal populations following expansion
with DCs.
In summary, polyclonal naturally occurring CD25+CD4+
Tregs only weakly suppress the allogeneic response, but the
culture of polyclonal CD25+CD4+ Tregs with allogeneic
DCs plus IL-2 both expands their numbers and leads to
potent alloantigen-specific suppressive activity both in vitro
and in vivo.
Expansion of alloantigen specific CD25+CD4+ Tregs by
manipulation of graft recipients
Another way to induce alloantigen-specific Tregs involves the
in vivo manipulation of recipients. Several methods for inducing transplantation tolerance in vivo are now known to generate antigen-specific CD25+CD4+ Tregs (reviewed in 15).
For example, alloantigen-specific CD25+CD4+ Tregs are
detected in mice that are tolerant to skin or cardiac grafts as
a result of donor-specific transfusion (DST) plus monoclonal
antibodies such as anti-CD154 (CD40-ligand), anti-CD4, or
anti-CD8 (15). CD25+CD4+ T cells from mice that had been
treated with anti-CD4 (YTS177) plus DST suppress skin graft
rejection with antigen specificity (77, 78). Similarly,
CD25+CD4+ T cells from mice tolerized with anti-CD154
inhibited allogeneic cardiac graft rejection mediated by
CD154-resistant CD8+ T cells in an antigen-specific manner
(79). It is also reported that CD25+CD4+ Tregs from antiCD154 antibody plus DST suppressed skin graft rejection
more potently than Tregs from naı̈ve mice (80, 81). These
observations might involve presentation of alloantigens by
DCs to CD25+CD4+ Tregs under circumstances where the
reactions of effector T cells are blocked but not totally, allowing for some IL-2 production.
There is a substantial difference in the Tregs that are
induced by alloDCs in vitro in the MLR and the Tregs that are
induced by DST. In vitro, DCs are primarily presenting intact
MHC molecules in the direct pathway of alloantigen presentation. During DST, there is evidence for an indirect pathway in
which donor MHC molecules are processed and presented by
host-derived APCs (82, 83). DCs have been shown to be
effective in processing dying allogeneic cells and presenting
antigens via the indirect pathway (84), but further experiments are required to directly establish a role for DCs in vivo in
inducing antigen-specific Tregs during DST combined with
blocking monoclonal antibodies.
Discussion
A good deal of the literature on CD25+CD4+ Tregs has
involved antigen-non-specific approaches. Bulk populations
of CD25+CD4+ Tregs are used to suppress autoimmunity (8,
9, 85) or inflammatory bowel disease (5, 41, 86) while
removal of CD25+CD4+ Tregs leads to enhanced resistance
to tumors (10, 87) or to increased responses to foreign
antigens (88, 89). However, there is evidence in autoimmune
disease that organ-specific Tregs are important for regulating
autoimmune thyroiditis, diabetes, encephalomyelitis, and
oophoritis (reviewed in 29). Likewise, during transplantation
tolerance following DST, antigen-specific Tregs are again
observed (15).
By studying the APC requirements for Tregs in culture, the
importance of antigen specificity in CD25+CD4+ Tregs function is quite clear. Tregs have long been regarded to be
anergic in vitro, but mature antigen-bearing DCs are able to
expand the numbers of Tregs. On a per cell basis, the
expanded populations then serve as more efficient suppressors
of responses by the corresponding antigen-reactive effector
cells. Here, we have emphasized studies in the allogeneic
MLR, which is the first naı̈ve polyclonal system used to
study the subject of antigen specificity. Exposure of purified
CD25+ CD4+ Tregs to allogeneic DCs from strain ‘A’ mice, in
the presence of exogenous IL-2, expands the number of cells
three to fivefold in a week. On a per cell basis, the expanded
Tregs are approximately 10-fold more effective in suppressing
a fresh MLR by CD25–CD4+ T cells to strain ‘A’ rather than
syngeneic or third party strain ‘B’ stimulators (Fig. 6A).
Likewise, the expanded Tregs suppress GVHD in vivo in a
strain-specific manner. In contrast, freshly isolated
CD25+CD4+ Tregs are much weaker in suppressing an MLR,
and there is no antigen specificity, i.e. Tregs from C57Bl/6
mice suppress the MLR comparably to BALB/c H-2d, CBA H2k, and SJL H-2s mice. Therefore, there is a strong antigenspecific component to Treg function that can be revealed by
expansion with antigen-presenting DCs (39).
Immunological Reviews 212/2006
325
Yamazaki et al Dendritic cells expand antigen-specific Tregs
Our studies have emphasized starting populations of peripheral Foxp3+CD25+CD4+ Tregs. In these experiments,
mature DCs expand total Treg numbers, suppressive function
on a per cell basis, and also the level of Foxp3 protein per cell.
Yet once the Tregs have been expanded by mature DCs,
suppressive function must be tested using APCs that contain
immature or maturing DCs, particularly within mouse spleen
cells. When mouse spleen cells are placed in culture, the DCs
begin to mature ‘spontaneously’, and one of the components
of maturation, the upregulation of CD80 and CD86 molecules, is blocked when CD25+CD4+ Tregs are added to the
culture. Other components, like the upregulation of MHC
class II, do not seem to be blocked. We suggest that during
any immune response, DCs will mature and induce IL-2 from
CD25–CD4+ T cells as well as the expansion of Tregs specific
for antigens on the mature DCs. The expanded Tregs will then
limit further expansion of Tregs from maturing DCs.
There are additional levels at which DCs can influence the
function of Tregs. DCs can expand and differentiate
Foxp3+CD25+CD4+ Tregs from Foxp3–CD25–CD4+ thymocytes, presumably with specificity for the self-antigens presented by thymic medullary DCs maturing in response to
thymic stromal lymphopoietin (90). In vivo, repeated injection
of preprocessed peptides (91) or the targeting of intact antigens to immature DCs (70) can convert antigen-specific
Foxp3–CD25–CD4+ T cells to Foxp3+CD25+CD4+ Tregs
bearing the same TCR. DCs also induce other regulatory
populations, such as IL-10-producing Tr1 (92–95) and
IL-10-producing regulatory DCs (96). The type of DC
population and/or DC maturation stimuli is different in
these systems, and select DC subsets may prove to be more
significant than others. Nevertheless, TGF-b production (69)
and IL-10 production (92, 93) by the antigen-presenting DCs
seem critical in at least some cases.
There are also two known antigen-non-specific components that are apparent with the studies of DC-expanded
Tregs. The first is that Tregs of one specificity can reduce
immune responses to other specificities, as long as the TCR
on the Treg is engaged. For example, Tregs specific for one
islet b cell autoantigen can reduce autoimmunity to multiple b
cell antigens (28), and Tregs specific for HA can block effector
T-cell responses to OVA (Fig. 1). This ‘non-specificity’ presumably results from the capacity of Tregs to dampen the function
of maturing DCs presenting different kinds of MHC-peptide
complexes, both MHC class I and II determinants. A second
type of non-specificity is evident in culture. When one adds
mature DCs to CD25+CD4+ TCR transgenic T cells, the latter
expand in the presence of IL-2 without the need to add the
corresponding peptide (although the combination of peptide
plus IL-2 leads to more T-cell expansion). Likewise in the MLR,
syngeneic DCs expand CD25+CD4+ T cells, and these express
Foxp3, but the expanded Tregs do not inhibit an alloMLR. In
both these examples, mature DCs plus IL-2 do not expand
CD25–CD4+ T cells in the absence of antigen (21). Therefore,
maturing DCs in the presence of IL-2 have some non-specific
capacity to expand preexisting Tregs in culture.
The MLR system might be useful in studying the APC requirements for human Tregs as well as the function of human Tregs
in disease. However, the experimental systems for studying
human Tregs are at the moment more demanding. In the
mouse, selection of CD25+CD4+ T cells with anti-CD25 antibody provides a population that is almost entirely Foxp3+, but in
humans, selection with CD25 yields mixed populations of
Foxp3+ and Foxp3– cells at least from the blood (97). The
expansion of functional human CD25+ CD4+ Tregs has been
achieved with anti-CD3 in the presence of allogeneic feeder
blood cells and IL-2 (98, 99) or anti-CD3 plus anti-CD28 in
the presence of IL-2 (100, 101). However, the polyclonally
expanded human Tregs were antigen non-specific in their suppressive function (99–101). Even human Tregs expanded by
allopeptide-pulsed immature DCs in the presence of IL-2 and IL7 showed antigen-non-specific suppression in vitro (102). The
problem of antigen-specific human Tregs is a challenging but
important one to resolve, since powerful approaches to immunotherapy could emerge if Tregs could be expanded that are for
example specific for b cell antigens in type 1 diabetes, intestinal
antigens in chronic inflammatory bowel disease, allergens in
asthma, and transplantation antigens in GVHD.
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