B7H1-Ig Fusion Protein Activates the CD4?IFN-? Receptor?
Type 1 T Regulatory Subset through IFN-?-Secreting Th1
Qing Ding,2* Liming Lu,2* Baolong Wang,* Yun Zhou,*†Yang Jiang,* Xiaorong Zhou,*
Lijun Xin,* Zhijun Jiao,* and Kuang-Yen Chou3*†
It has been demonstrated in our previous work that, in the human skin-grafting model, the expression of costimulatory molecule
B7H1 (PD-L1) by keratinocytes plays an essential role in inducing local tolerance via activation of IL-10-secreting T cells. This
study further analyzes the role of B7H1 in differentiation of type 1 T regulatory (Tr1) cells and explores underlying mechanisms.
Mouse fusion protein B7H1-Ig is used, together with immobilized anti-CD3 mAb, to costimulate the purified naive CD4?T cells.
B7H1-Ig-treated CD4?T cells were found to activate a characteristic Tr1 population possessing a CD4?CD25?Foxp3?
CD45RBlowphenotype. These regulatory T cells strongly inhibited the Th1-dominated MLR by secretion of IL-10 and TGF-?.
Moreover, B7H1-treated Tr1 cells also resulted in suppressed clinical scores and demyelination when adoptively transferred into
mice with experimental allergic encephalomyelitis. Furthermore, analysis of the cytokine profile indicated that there were two
differential reaction patterns during the B7H1-Ig-induced Tr1 development. These two patterns were characterized by activation
of IFN-?R?IL-10R?Th1 and IFN-?R?IL-10R?Tr1 cells, respectively. Secretion of IFN-? by Th1 and the expression of IFN-?R
on Tr1 were critical for further Tr1 differentiation, as demonstrated by mAb blocking and by analysis in IFN-??/?mice. In
conclusion, B7H1 is capable of inducing Tr1 differentiation from naive CD4?T cells by coactivation in an IFN-?- or Th1-
dependent manner. Our study may shed some light upon the clinical usage of B7H1 as a therapeutic reagent for induction of
tolerance. The Journal of Immunology, 2006, 177: 3606–3614.
disease (1–3). Two main categories of Treg cells, natural and adap-
tive, have been proposed (3–5). As representatives of these,
CD4?CD25?T and the type 1 Treg (Tr1) cells have received great
attention in recent years. Treg cells are believed to be active in
autoimmune disease (6, 7), tumorigenesis (8–11), transplantation
tolerance (12, 13), allergic disease (14–17), infection (18–22), oral
tolerance (23), and the maternal-fetal relationship (24).
The naturally occurring CD4?CD25?T cells differentiate in the
thymus and emerge into peripheral tissues, where they suppress the
activation of self-reactive T cells in a basically cell contact-depen-
dent manner (1–5). Their development is characterized by the in-
egulatory T (Treg)4cells have become a central topic in
basic and clinic immunology owing to their role in main-
tenance of homeostasis and in pathogenesis of clinical
fluence of transcription factor Foxp3, also leading to a constitutive
expression of Foxp3 as a new surface marker (25, 26). The
CD4?CD25?Foxp3?Treg cells are thus distinguishable from the
CD25-inducibly expressed, Ag-activated T cells that possess a
CD4?CD25?Foxp3?phenotype. In contrast, Tr1 cells arise in the
periphery upon encountering Ags in the presence of exogenous
IL-10. The unique cytokine profile of IL-2?IL-4?IL-10?TGF-??
places Tr1 cells into a subset distinct from those of Th0, Th1, or
Th2 cells (27–31). This is despite the fact that Tr1 cells also pro-
duce IFN-? at levels lower than that produced by Th1 cells. There
is still intensive interest in better defining the origins, develop-
ment, phenotype, and potential clinical application of these cells.
An active role for dendritic cells (DCs), for example, has been
demonstrated in Tr1 activation (32–35). G-CSF, it is also reported,
can induce Tr1 cells via IL-10 and IFN-? (36, 37). Stimulation of
T cells in the presence of immunosuppressant such as vitamin D3
and dexamethasone has a similar Tr1-inductive effect (38, 39).
However, little is known about the role of costimulatory molecules
and their possible interactions with functional T subsets in Tr1
In the 1980s, thousands of severe burn patients were success-
fully rescued in China by treatment using intermingled skin graft-
ing (40, 41). The grafted skin was elaborated by creating a mosaic
of allogeneic skin sheets imbued with many tiny autoskin pieces.
The presence of the autoskin (skin islets) in the transplant could
induce strong local tolerance to protect the intermingled skin from
rejection, which was helpful in preventing infection. Indeed, we
were able to show that the keratinocytes, a type of nonprofessional
APC within the autoskin pieces, could be activated to express the
B7H1 molecule. This, in turn, activated IL-10-secreting cells to
create a local environment of immune low responsiveness (42).
*Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medi-
cine, Shanghai, China; and†E-Institute of Shanghai Universities, Immunology Divi-
sion, Shanghai, China
Received for publication March 16, 2006. Accepted for publication June 26, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by grants from the Natural Science Foundation of China
(30530690), the National Key Basic Research Program of China (2003AA205009,
2001CB 510003), and the Foundation of Shanghai Sci-Tech Council (03JC14085,
2Q.D. and L.L. contributed equally to this work as first authors.
3Address correspondence and reprint requests to Dr. Kuang-Yen Chou, Shanghai
Institute of Immunology, Shanghai Jiaotong University School of Medicine, 280
South Chongqing Road, Shanghai 200025, China. E-mail address: email@example.com
4Abbreviations used in this paper: Treg, T regulatory; Tr1, type 1 Treg; DC, dendritic
cell; EAE, experimental allergic encephalomyelitis; LN, lymph node; MOG, myelin
oligodendrocyte glycoprotein; Nrp1, Neuropilin-1; RR, relative response; rmIL-2,
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
Moreover, blocking of B7H1 with a mAb completely abrogated
the ability of the autokeratinocytes to induce tolerance.
B7H1 (PD-L1), a member of the B7 costimulatory family ca-
pable of costimulating T cells to proliferate and secrete IL-10, was
first reported in humans by Dong et al. (43) and Freeman et al.
(44). It was further demonstrated that the down-regulatory activity
exerted by B7H1-activated T cells was dependent, not only upon
IL-10 production, but also upon PD-1, a specific receptor bearing
the ITIM. No reports to date, however, deal with the role of B7H1
in induction of Tr1.
Based upon our work on transplantation tolerance as induced by
B7H1-expressed keratinocytes in the intermingled skin graft, we
undertook to examine the role of B7H1 in induction of Tr1 and
associated underlying mechanisms in an APC-absent system. Pu-
rified mouse naive CD4?T cells were stimulated with anti-CD3
mAb and mouse B7H1-Ig fusion protein. In this way, the costimu-
latory molecule B7H1, rather than anti-CD28 Ab, was able to in-
duce Tr1 cells. IFN-? and IFN-?-secreting Th1 cells were seen to
be critical for the differentiation of Tr1. A Tr1 population express-
ingIFN-?R was accordingly
CD4?Foxp3?CD45RBlowIFN-?R?IL-10R?. Thus, for the first
time, activation of Tr1 cells can be seen to be directly costimulated
by B7H1 in the presence of Th1-secreted IFN-?.
Materials and Methods
Isolation of mouse naive T cells and fractionation of T subsets
C57BL/6 mice were originally obtained from The Jackson Laboratory and
maintained at Sino-British Sippr/Bk Laboratory Animal. Our study was
approved by the Scientific Investigation Board of Shanghai Jiaotong Uni-
versity School of Medicine. Mouse naive CD4?T cells were isolated from
splenocytes by using nylon wool column (Wako Chemicals) and MACS
(Miltenyi Biotec). After checking for phenotype as CD4?CD62L?by flow
cytometry, the naive T cells were further fractionated using MACS with
anti-CD25-coupled magnetic beads to obtain the CD4?CD25?T and
CD4?CD25?T subsets. The purity of isolated T cells and their subsets
reached ?95%, as determined by flow cytometry.
Activation of naive T cells with anti-CD3 plus costimulators
The naive CD4?T cells were stimulated in vitro with anti-CD3 mAb
together with one of the following costimulators: mouse fusion protein
B7H1-Ig (R&D Systems) (45), anti-CD28 mAb (BD Pharmingen), or un-
related Ig (including mouse IgG2a; R&D Systems) as an unrelated Ab for
negative control. Before the naive T cells were seeded at a concentration of
2 ? 106cells/ml on culture plates, the anti-CD3 mAb (200 ng/ml; BD
Pharmingen) was precoated with one of the costimulators on 96-well flat-
bottom plates (Costar). Coating proceeded overnight to produce three com-
binations: anti-CD3 plus B7H1-Ig (5 ?g/ml), anti-CD3 plus anti-CD28 (2.5
?g/ml), and anti-CD3 plus Ig-control (5 ?g/ml). The activated cells were
thus designated T-B7H1, T-CD28, and T-control, respectively.
T cell proliferation
Lymphoproliferation was assayed by adding [3H]TdR (sp. act., 32 Ci/mM;
Shanghai Institute of Atomic Nucleus, Chinese Academy of Sciences) at 1
?Ci/well 16 h before termination of culture. Isotope incorporation was
determined with a liquid scintillation counter (MicroBeta TriLux). Results
are expressed either as mean ? SD of cpm for triplicates/quadruplicates or
as relative response (RR). RR (percentage) ? (cpm of experimental com-
bination/cpm of control) ? 100.
In some cultures, anti-IL-10, anti-TGF-?, and anti-IFN-? (R&D Systems)
or anti-PD-1 (eBioscience) mAbs were added separately at concentration
of 20 ?g/ml.
Double stimulation of CD4?T or CD4?CD25?T cells with
anti-CD3 plus B7H1-Ig
To enrich the Tr1 cell population, achieving higher purity and concentra-
tion, the naive CD4?T cells were twice stimulated with anti-CD3 plus
costimulator. The primary stimulation was conducted in 24-well culture
plates (Costar) on which anti-CD3 mAb, B7H1-Ig, or Ig-control was pre-
coated, as described above, and the T cells were added at 2 ? 106cells in
1 ml of culture medium/well. Three days later, 1 ml of mouse rIL-2
(rmIL-2) (20 ng/ml; R&D Systems) was added to allow cell expansion. On
day 7, the cells were collected, washed, and restimulated with the same
stimuli under the same conditions. The resulting cells were washed to
remove residual rmIL-2. Those twice stimulated with anti-CD3 plus
B7H1-Ig are referred to as T-(B7H1) to distinguish them from the T-B7H1
that were activated only once, and those twice stimulated with anti-CD3
plus Ig-control are designated as T-(control). As mentioned above, when
anti-IL-10R, anti-TGF-?, or anti-IFN-?R (R&D Systems) mAbs were
added into the culture on day 0 together with anti-CD3 plus costimulators,
the resultant twice-stimulated cells are referred as, for example, T-(B7H1 ?
anti-IL-10R) or T-(B7H1 ? anti-IFN-?R).
Suppressive activity of twice-stimulated CD4?T cells
To test suppressive capacity, an MLC was established with syngeneic
CD4?T cells of C57BL/6 origin as responders and 6000-rad irradiated
BALB/c (H-2d) (Sino-British Sippr/Bk Laboratory Animal) CD3-depleted
splenocytes as stimulators. Dosage of both responders and stimulators in
MLC was 5 ? 105per well. T-(B7H1) or T-(control) cells were added to the
MLC at the same concentration in 200 ?l of complete medium in 96-well
round-bottom plates (Costar). In some experimental combinations, anti-
IL-10 (20 ?g/ml) and/or anti-TGF-? (20 ?g/ml) mAbs were added to the
MLC. Lymphoproliferation was assayed with [3H]TdR by the procedure
described above. Percentage of suppression is calculated as 100 ? RR.
Induction of experimental allergic encephalomyelitis (EAE) by
myelin oligodendrocyte glycoprotein (MOG)35–55
To induce EAE, female C57BL/6 mice (6–10 wk old) were immunized by
s.c. injection in three sites on the flank with a total of 200 ?g of antigenic
peptide MOG35-C55(MEVGWYRSPFSRVVHLYRNGK) (46) (synthe-
sized by GL Biochem) in 200 ?l of CFA containing 800 ?g of Mycobac-
terium tuberculosis H37Ra (Difco). On days 0 and 2, each mouse received
an additional 200 ng of pertussis toxin (Sigma-Aldrich) in 200 ?l of PBS.
Evaluation of clinical scores was done using a scale of 0–5 in a blinded
manner according to Tompkins et al. (47): 0, no abnormality; 1, limp tail;
2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb
paralysis and forelimb weakness; 5, moribund. Mean daily clinical scores
were recorded and compared with age- and sex-matched controls.
For initiation of EAE by adoptive transfer of sensitized T cells, draining
lymph nodes (LN) were collected from mice that had been challenged with
peptide MOG35-C55for 7–11 days. LN cells were cultured (107/ml) in
RPMI 1640 complete medium with the peptide (40 ?g/ml) and rmIL-2 (20
ng/ml) for 72 h. The MOG35-C55-sensitized T cells were harvested, washed,
and resuspended at 5 ? 107/ml in RPMI 1640. On day 0, the cells were
injected i.v. into naive C56BL/6 mice at a dose of 5 ? 107cells per in-
jection. The mice were treated with 200 ng of pertussis toxin in 200 ?l of
PBS on days 0 and 2 (48).
Assessment of the effects of T-(B7H1) upon MOG peptide-induced
MOG35–55-sensitized CD4?T cells were isolated using MACS from LN
cells of EAE mice. The cells were cocultured for 72 h at 5 ? 105/well with
increasing numbers of T-(B7H1) or T-(control) cells, together with 4000-
rad irradiated syngeneic CD3?splenocytes (5 ? 106/well) and the peptide
MOG35–55(40 ?g/ml). The suppressive effects of T-(B7H1) on the second
response of sensitized T cells to peptide MOG35–55were assessed by de-
termination of lymphoproliferation.
Two protocols were adopted to evaluate the effects of T-(B7H1) on EAE
in vivo. First, 5 ? 107of T-(B7H1) or T-(control) cells was transferred i.v.
into mouse recipients 3 days before EAE was actively induced with peptide
MOG35–55plus CFA. Second, EAE was adoptively induced in mice by
cotransferring the MOG35–55-sensitized lymphoblasts and T-(B7H1) or T-
(control) cells on day 0. To assess the effects of T-(B7H1) in vivo, clinical
scores were recorded and spinal cords were collected and fixed in 10%
phosphate-buffered Formalin for histopathological analysis. The paraffin-
embedded tissue sections were stained either with H&E or with Luxol fast
blue to examine infiltration of mononuclear cells or demyelination,
Real-time quantitative PCR
Foxp3 and Neuropilin-1 (Nrp1) have been recently identified as specific
markers for CD4?CD25?Treg (25, 26, 49). It has also been reported that
chemokine receptor CCR5 and transcription factor T-bet are predominantly
expressed in Th1 cells (50, 51), and that CCR3 and GATA3 are restricted
to expression in Th2 cells (51, 52). To quantitatively determine levels of
Foxp3, Nrp1, IL-10, IFN-?, CCR5, CCR3, T-bet, GATA3, IFN-?R, and
IL-10R at mRNA level, real-time PCR was performed using LightCycler
3607 The Journal of Immunology
RNA Master SYBR Green I Kit (Roche Applied Science) with the follow-
ing primers: Foxp3, sense ATTGGTTTACTCGCATGTTCG and antisense
GTCAAGGGCAGGGATTGG; Nrp1, sense TCACATTGGGCGTTA
TTG and antisense CACTGTAGTTGGCTGAGAAA; IL-10, sense ACC
TGGTAGAAGTGATGCC and antisense CACCTTGGTCTTGGAGCT;
IFN-?, sense TGAGACAATGAACGCTACA and antisense TTCCACAT
CTATGCCACT; CCR5, sense CTGAAGAGCGTGACTGAT and anti-
sense-ACATTATGTTCCCAAAGAC; CCR3, sense TCTCCTGAGAT
GTCCCAATA and antisense TCACCAACAAAGGCGTAG; IL-10R,
sense ACATTCGGAGTGGGTCAA and antisense GAGAAACGCAG
GTGTAAAG; and IFN-?R, sense TCCTACATACGAAACATACGG and
Samples were run in duplicate, and expression levels were normalized to
expression of hypoxanthine phosphoribosyltransferase in each set of sam-
ples to calculate the degree of change.
Determination of cytokine production
For cytokine analysis, cell cultures were set up as described above and
supernatants were collected at indicated times and stored at ?20°C. Su-
pernatant concentrations of IL-2, IL-4, IL-10, IL-12, IFN-?, and TGF-?
were determined using cytokine ELISA kits (Bender MedSystems). Results
were determined on a MR7000 plate reader (Dynatech Laboratories) at 450
nm and analyzed using BIOLINX software.
Percentages of cells with positive surface markers labeled with fluorescent
dye-conjugated mAbs were assayed using flow cytometry. For example,
identification of two CD4?populations with or without IL-10R and
IFN-?R was accomplished using FITC-conjugated anti-biotin (Sigma-Al-
drich), biotin-conjugated anti-IFN-?R (BD Pharmingen), and PE-conju-
gated anti-IL-10R (BD Pharmingen) mAbs. Analysis was performed on
FACSCalibur (BD Biosciences) using CellQuest software.
A Student’s paired t test was used to determine significant differences (p ?
0.05). Error bars in figures represent the SD of triplicates or quadruplicates
for cell cultures.
Differential reaction patterns in early and late course of CD4?
T cell response to B7H1-Ig and TCR stimulation
In our previous work, IFN-?-induced B7H1?keratinocytes and
B7H1-transfected mouse endothelial cells were successfully used
to induce IL-10-secreting T cells (42). To exclude the possibility of
interference from accessory cells, highly purified mouse naive
CD4?CD62L?T cells were used in this study and were stimulated
with anti-CD3 mAb plus fusion protein B7H1-Ig. Anti-CD28 mAb
and unrelated Ig-control were used as positive and negative con-
trols in the presence of anti-CD3 mAb. Fig. 1A shows two distin-
guishable reaction patterns of lymphoproliferation during a 6-day
period. The early phase (day 1–3) was characterized by a rapid
synchronous proliferation of the CD4?T cells for all three kinds
of costimulatory molecule, especially for B7H1-Ig and anti-CD28.
During the later phase (day 4–6), however, reaction patterns di-
versified. After costimulation with anti-CD28, for example, the
intensity of lymphoproliferation was maintained. In contrast, when
costimulated with B7H1-Ig, the [3H]TdR uptake for T cell prolif-
eration dropped sharply on day 4 and continued to decrease. This
indicated that the B7H1-Ig-costimulated CD4?T cells lost their
proliferative capacity during this phase.
To confirm the differential effects of the costimulatory B7H1-Ig
in the two phases, we examined the dose-effect curves on days 3
and 6, respectively. As indicated in Fig. 1, B and C, the enhance-
ment and depression patterns of lymphoproliferation reversed,
while the response levels in the control (treated with Ig-control)
remained basically unchanged. The cpm value for lymphoprolif-
eration on day 6, for example, dropped to 75% suppression when
the dose of B7H1-Ig was increased to 10 ?g/ml. These results
suggest that some CD4?T cell-derived components (cells and/or
soluble factors) with inhibitory activity began to emerge by day 4
of lymphoproliferation and reached their maximum levels by the
To examine the cytokine profiles, three kinds of costimulators
were tested separately. When CD4?T cells were treated with anti-
CD3 plus anti-CD28, there ensued an enhanced secretion of typical
Th1-related cytokines IFN-? and IL-2 (Fig. 2, A and B) rather than
IL-10, TGF-?, and IL-4 (Fig. 2, C–E). In contrast, when anti-
CD28 was replaced by B7H1-Ig, the secretion levels of IL-10 and
TGF-? were dramatically increased during the later phase (day
4–6), rapidly reaching high levels (Fig. 2, C and D). It is of in-
terest to note that B7H1-Ig was also capable of stimulating some
production of IFN-? on days 2 and 3 (Fig. 2B). On day 4, its
concentration decreased to a moderate level, although the secretion
of IL-2 never reached a significant amount (Fig. 2A). Fig. 2E in-
dicates that T cells costimulated either with anti-CD28 or with
B7H1-Ig produced little detectable IL-4, suggesting no involve-
ment of a Th2 subset in the response. The high levels of IL-10 and
TGF-? that were detectable only for the B7H1-Ig-costimulated
CD4?T cells strongly implicate another T cell subset, Tr1, as
being activated in the later phase.
IFN-? is critical for IL-10 production by B7H1-Ig-costimulated
The mAb-blocking experiment further indicated that the suppres-
sion induced by B7H1-Ig-costimulated CD4?T cells could be
partially diminished by adding mAbs against IL-10, TGF-?, or
PD-1, respectively (Fig. 3A). Among them, anti-IL-10 had the
strongest blocking activity. PD-1 is an ITIM-bearing inhibitory
receptor capable of inducing suppression or apoptosis when ligated
with the B7H1 molecule (44, 53). Some reverse effects of sup-
pression were also observed when anti-PD-1 mAb was used (p ?
0.05, Student’s t test). The combination of these three mAbs
yielded the strongest recovery from B7H1-Ig-induced suppression,
suggesting that, besides cytokines IL-10 and TGF-?, the effective
eration. A, Differential time proliferation curves showing two-phase pat-
terns for the lymphoproliferation in which purified naive CD4?T cells
were stimulated by immobilized anti-CD3 mAb together with one of the
following costimulators: B7H1-Ig fusion protein, anti-CD28 mAb, or Ig
control. The results are representative of four independent experiments. B
and C, Dose-effect curves determined on days 3 and 6 of T lymphoprolif-
eration, respectively, which were stimulated with anti-CD3 and B7H1-Ig.
Influences of the fusion protein B7H1-Ig on lymphoprolif-
3608B7H1-INDUCED Tr1 DIFFERENTIATION DEPENDS ON IFN-?
function of PD-1 might also contribute to the hyporeaction during
the later phase. This could be further verified by the recovery pat-
tern that did not reach its maximum when only anti-IL-10 and
anti-TGF-? were combined together (Fig. 3A).
Quite interestingly, anti-IFN-? mAb could not diminish the
B7H1-Ig-induced suppression. Instead, the intensity of lympho-
proliferation was weakened somehow (Fig. 3A), suggesting that
the IFN-? might exert its role on Tr1 differentiation instead as an
effective factor for B7H1-Ig-induced suppression. This stimulated
our interests for further exploration. When IL-10 was taken into
account as effective molecule, for example, it was revealed that the
production of IL-10 by the B7H1-Ig-costimulated T cells was in-
creased and much earlier when exogenous IFN-? was added into
culture at day 0 (Fig. 3B). Both anti-IFN-? and anti-IFN-?R mAbs
were able to suppress IL-10 production dramatically. There was no
effect detected, however, in the presence of exogenous IFN-? in
the Ig-control-costimulated CD4?T cells (Fig. 3B).
When CD4?T cells were isolated from IFN-? gene knockout
mice (IFN-??/?) (originally from The Jackson Laboratory) and
assayed by intracellular staining for IL-10-producing cells (IL-10?
T) after treatment with anti-CD3 plus B7H1-Ig, the percentage of
IL-10?T cells was reduced by 84.7% (i.e., from 17.0% for wild-
type mice to 2.6% for IFN-??/?mice). Adding back IFN-? could
help restore the percentage of IL-10?T cells to 20% (Fig. 3C).
Identification of two CD4?T populations during the response to
anti-CD3 plus B7H1-Ig
Several important results were observed when cell surface markers
and the Th1/Th2-specific transcription factors T-bet/Gata3 were
examined using real-time PCR. 1) As expected, for anti-CD28 co-
stimulation, the expression of Th1-related CCR5 molecule and T-
bet was greatly increased in CD4?T cells from day 2 to day 6
(Fig. 4, A and B). In contrast, costimulation with B7H1-Ig induced
the expression of CCR5 and T-bet at only a moderate level during
the early phase. It is notable that expression was rapidly depressed
during the later phase. 2) Compared with the negative control (co-
stimulated with Ig-control), expression of IL-10R at very high lev-
els could be seen during the later phase when CD4?T cells were
costimulated with B7H1-Ig instead of with anti-CD28 (Fig. 4C). 3)
Both CD28- and B7H1-Ig-costimulated T cells were able to ex-
press more IFN-?R molecule as compared with baseline levels
(Fig. 4D). 4) No CCR3?Gata3?T cells were detectable (Fig. 4, C
and D) for the three costimulators evaluated, confirming again that
a Th2 subset was not involved in the Tr1-related suppression.
These results, together with the proliferation patterns and the
cytokine profiles depicted in Figs. 2 and 3, strongly suggest that,
for costimulation with B7H1-Ig, a Th1 (or a Th1-like) subset was
activated during the early phase, followed by differentiation into an
IL-10-secreting T subset with suppressive properties.
To confirm this, IL-10R and IFN-?R were invoked as two pa-
rameters with which to define the two populations by flow cytom-
etry in the 6-day course of B7H1-Ig-costimulated lymphoprolif-
eration. As indicated in Fig. 5, the first population emerging on day
2–4 was phenotypically IFN-??IL-10R?. The other population,
characterized as IFN-??IL-10R?, appeared later and began to be
emerged on day 3 (Fig. 5E). Its proportion could gradually reach
93.4% on day 6 from 11.6% on day 3 and 76.1% on day 5 (Fig.
5G), accompanied by a decrease in the first population from 72.8%
on day 3 to 2.7% on day 6. In contrast, when anti-CD28 was used
as costimulator, the IFN-??IL-10R?T population kept expanding
on a very high level (?90%) from day 1 to day 5 with no activa-
tion and expansion of IFN-??IL-10R?T cells (Fig. 5, J–N).
Characterization of T-(B7H1), the CD4?T cells costimulated
twice with B7H1-Ig
As indicated above, most of the CD4?T cells stimulated with
anti-CD3 plus B7H1-Ig developed into a subset with down-regu-
latory ability by day 6. To further purify and enrich the subset, the
CD4?T cells primarily activated for 3 days with anti-CD3 plus
B7H1-Ig were expanded with rmIL-2 for another 4 days and then
restimulated with anti-CD3 plus B7H1-Ig for an additional 6 days.
The resultant subset (cultured for 14 days total) is designated as
T-(B7H1) to distinguish it from T-B7H1 costimulated once
with B7H1-Ig for 6 days. As indicated in Fig. 5H, the
costimulation with anti-CD28, there appeared a Th1-related cytokine profile (A and B). After costimulation with B7H1-Ig, high levels of IL-10 and TGF-?
were detected during the later phase of the response (C and D). No Th2-related IL-4 was detectable for all experimental combinations (E). The levels of
cytokines were quantitatively determined by ELISA as ng/ml in culture supernatants.
Cytokine secretion patterns of the CD4?T cells costimulated with unrelated Ig, anti-CD28, or B7H1-Ig in presence of anti-CD3 mAb. For
3609The Journal of Immunology
IL-10R?IFN-?R?T-(B7H1) could reach a purity as high as 98.5%.
In contrast, the histogram patterns of the T-(control) remained un-
changed either for single (6 days) or double (14 days) treatment
(Fig. 5, O and P) in comparison with the T cells receiving no
treatment (Fig. 5, A and I). Proliferation assays (Fig. 6A) verified
that IL-10 secretion by T-(B7H1) was much greater and peaked
earlier than that of T-B7H1. Functionally, T-(B7H1) was able to exert
strong suppression on MLC in which syngeneic CD4?T cells
were set to respond to allogeneic splenocytes, although the
T-(B7H1) themselves were not able to proliferate to the allogeneic
stimulation (Fig. 6A).
In contrast to the Th1-related cytokine profile seen in the control
group (Fig. 6C), the suppression of T-(B7H1) cells on the MLC was
correlated to their high levels of IL-10 and TGF-? secretion (Fig.
6B). It is also notable that production of IL-4 remained at very low
levels in experiments using either T-(B7H1) or T-(control) (Fig. 6,
B and C).
To explore the possible connection with CD4?CD25?T cells,
CD4?T cells were fractionated into CD25-positive and CD25-
negative subsets and two CD4?CD25?Treg-specific markers,
Foxp3 and Nrp1, were assayed using real-time PCR. As indicated
in Fig. 7A, the markers were not detected in CD4?CD25?T cells
either before or after treatment with anti-CD3 plus qB7H1-Ig. For
total T cells, the relative expressions of the two markers remained
unchanged during costimulation with B7H1-Ig. This implies that
the B7H1-Ig-costimulated T-(B7H1) cells might be irrelevant to the
lineage of CD4?CD25?Foxp3?Treg cells. Functional analysis
further supported this point because both T-(B7H1) and
CD4?CD25?T-(B7H1) had similar capabilities for inducing strong
suppression on MLR (Fig. 7C). However, when CD45RB on T
cells was examined by flow cytometry before and after treatment
with anti-CD3 plus B7H1-Ig, its mean expression intensity (indi-
cated as Y-mean) shifted from 1236 to 462, or from CD45RBhigh
to CD45RBlow(Fig. 7B). CD45RBlowhas been reported as a
marker for Treg cells in mice (54, 55).
These results, together with the functional analysis and cytokine
profile examination, indicate that the T-(B7H1) belongs to
CD4?Foxp3?CD25?CD45RBlowTr1, which induces immune
suppression via secretion of IL-10 and TGF-?.
Ligation of IFN-?R and IL-10R is required for differentiation
To reveal the role of IFN-?R in Tr1-related suppression, anti-
IFN-?R and anti-IL-10R mAbs were added during the induction of
Ig-costimulated CD4?T cells. A, Blocking effects of lymphoproliferation
by mAbs. In contrast to diminishing effects of anti-IL-10, anti-TGF-?, and
anti-PD-1 mAbs on lymphoproliferation induced by anti-CD3 plus
B7H1-Ig (5 ?g/ml), anti-IFN-? mAb showed no effect (f) on day 6 when
Tr1 cells have completed their differentiation. Results are expressed as RR
in comparison with the proliferation of CD4?T cells stimulated with anti-
CD3 plus Ig control. Significant differences were detected between the
indicated experimental combinations by Student’s t test as ? (p ? 0.05) and
?? (p ? 0.01). B, Blocking effects of anti-IFN-?/IFN-?R mAbs on IL-10
production in the B7H1-Ig-costimulated CD4?T cells. C, A comparison of
IFN-? and IL-10 production in the B7H1-Ig-stimulated CD4?T cells that
were originally collected from regular or IFN-??/?mice. The depression
of the CD4?IL-10?T cells from 17.0% in regular C57BL/6 to 2.6% in
IFN-??/?mice can be reversed up to 20% when exogenous IFN-? was
added (see right below histogram). The IL-10?T cells were assayed by
intracellular staining with flow cytometry.
Involvement of IFN-? in induction of IL-10 by the B7H1-
by real-time PCR during 6-day culture of CD4?T cells stimulated with
anti-CD3 plus costimulators. A and B, When T cells were costimulated
with anti-CD28, there was a strong expression of Th1-related CCR5 and
T-bet. After costimulated with B7H1-Ig, however, CCR5 and T-bet were
expressed only in the early phase at a moderate level and rapidly decreased
or disappeared by the later phase. C, IL-10R was strongly expressed in the
later phase in a unique pattern when B7H1-Ig was used as costimulator. D,
The expression of IFN-?R in the T cells costimulated with either B7H1-Ig
or anti-CD28 is similar. E and F, No Th2-related CCR3 and Gata3 were
detectable for all costimulators in presence of anti-CD3.
Determination of surface markers and transcription factors
3610B7H1-INDUCED Tr1 DIFFERENTIATION DEPENDS ON IFN-?
T-(B7H1). Two distinct subsets emerged, designated T-(B7H1 ?
?IFN-?R) and T-(B7H1 ? ?IL-10R). In addition, the control subset
T-(B7H1 ? Ig-control) is noted. The results presented in Fig. 7D in-
dicate that either T-(B7H1) or T-(B7H1 ? Ig-control) could induce sup-
pression of MLR, as expected. However, the suppression was dra-
matically diminished when T-(B7H1 ? Ig-control) was replaced with
T-(B7H1 ? ?IFN-?R) or with T-(B7H1 ? ?IL-10R), strongly suggesting
that these two subsets were unable to induce suppression, or that
they no longer belonged to Tr1. The effects of perturbing Tr1 dif-
ferentiation using anti-IFN-?R and anti-IL-10R mAbs are similar
to those seen with the T-(B7H1) that were originally isolated from
IFN-??/?mice in our MLR testing system (Fig. 7C).
These results demonstrate that the ligation of IFN-?R with
IFN-? on the surface of the T-(B7H1) or T-(B7H1) precursors is
essential for differentiation and activation of Tr1. The Th1 subset,
from which the original IFN-? was produced, could function as a
kind of inducer for Tr1 differentiation.
Therapeutic activity of T-(B7H1) as Tr1 in treatment of EAE
To further confirm that the T-(B7H1) is functionally equal to Tr1, a
mouse model with EAE was used in immunization of C57BL/6
mice with autologous Ag-derived peptide MOG35-C55in adjuvant.
As seen in Fig. 8A, coculture of the MOG35–55-specific T cells in
fixed number with increasing numbers of T-(B7H1) cells inhibited
the T cell proliferation in a dose-dependent manner. In vivo ex-
periments further demonstrated that when the T-(B7H1) cells were
adoptively transferred into naive recipients 3 days before EAE was
actively induced, T-(B7H1) conferred significant protection from
the development of clinical EAE (Fig. 8B) in comparison with
mice receiving either no cells or T-(control) cells. In another adop-
tive transfer experiment, T-(B7H1) cells were coinjected with pre-
viously activated MOG35–55-specific T lymphoblasts, resulting in
53% reduction of clinical symptoms in comparison with mice re-
ceiving T-(control) cells plus T lymphoblasts (Fig. 8C). Thus, sup-
plementation with T-(B7H1) conferred protection against progres-
sion of both actively and passively induced EAE.
When spinal cord tissue was fixed and stained with H&E to
examine mononuclear cell infiltration characteristic of EAE dis-
ease progression in CNS inflammation, infiltration was substan-
tially reduced in the T-(B7H1)-treated recipients (Fig. 8D). This
correlated well with observed changes in disease severity. When
fixed spinal cord tissue was stained with Luxol fast blue, demy-
elination was seen to be substantially reduced in the T-(B7H1)-
treated group (Fig. 8E).
DCs, especially immature or semimature DCs, have been reported
to be capable of inducing Tr1 rather than CD4?CD25?Treg in
allergic diseases (32–35). It is thus believed that the activation
status of the DCs and IL-10 secretion by innate cells may be the
major influences on the induction of Tr1 cells (4). In addition to the
roles of DCs and exogenous IL-10, treatment with IFN-? or G-
CSF appeared to be effective in inducing Tr1 cells (36, 37). Stim-
ulation of T cells in the presence of vitamin D3 and dexametha-
sone had a similar Tr1-inducing effect, which depended on
induction of autocrine IL-10 (38). Interestingly, costimulation via
CD2 or with Abs against CD46 also resulted in the generation of
Tr1 cells. From these studies, conducted in the absence of APCs,
it is suggested that certain soluble factors might be involved in, or
anti-CD3 and B7H1-Ig (T-(B7H1)). A, A comparison of B7H1-Ig-costimulated
T cells with respect to primary (T-B7H1) and secondary (T-(B7H1)) re-
sponses. A, The suppressive activity of T-(B7H1) on lymphoproliferation
stimulated by allogeneic splenocytes. Differential profiles of cytokine pro-
duction are seen when naive CD4?T cells and allogeneic splenocytes in
MLC were cocultured with syngeneic T-(B7H1) (B) or T-(control) (C) (the
Ig-control twice-costimulated T cells).
Characterization of the CD4?T cells twice stimulated with
A, Negative control with no stimulation. B–D, During day 1–3, an IFN-?R?IL-10R?T subset was recorded at a ratio of 58.3:72.8%. D–G, Another T
population with phenotype IFN-?R?IL-10R?was activated from day 3 (11.6%) and reached its peak level at day 6 (93.4%), accompanied by the
disappearance of the former T cell subset that was declined from 72.8 to 2.7%. H, When CD4?T cells were costimulated twice with B7H1-Ig for 14 days,
the IFN-?R?IL-10R?T population achieved 98.5% purity. J–N, There were no similar IFN-?R?IL-10R?T cells detectable after CD4?T cells were
costimulated with anti-CD28 in comparison with that in control (I). O and P, Same was for costimulation with Ig control for 6 days (once stimulated) and
14 days (twice stimulated).
Differentiation of two cell populations during the response course when CD4?T cells were stimulated by anti-CD3 plus B7H1-Ig for 6 days.
3611 The Journal of Immunology
even sufficient for, the differentiation of Tr1 cells. In allergen-
induced airway hyperreactivity, for example, pulmonary DCs
could stimulate the development of Tr1 in a manner similar to
costimulation with ICOS ligand (17), a member of the B7 family.
Since we reported that B7H1-positive keratinocytes able to induce
local tolerance in the intermingled skin-grafting procedure via IL-
10-secreting T cells (42), there have been no investigations dealing
with the relationship of B7H1 and Tr1 activation. This study
MOG35–55-sensitized CD4?Th1 cells to MOG35–55in presence of irradiated autologous splenocytes. Lymphoproliferation is presented as cpm. B and C,
EAE regression in the C56BL/6 mice treated with T-(B7H1) cells. The cells were administrated either on day 3 before EAE was actively induced with
MOG35-C55on day 0 (B) or passively induced by simultaneous injection of T-(B7H1) plus the MOG35-C55-sensitized T lymphoblasts on day 0 (C). There
are significant differences (p ? 0.01) between the experimental group (with T-(B7H1)) and the controls (with T-(control) or no T cells) in terms of mean
clinical scores (n ? 5 in each group). D and E, Demyelination and inflammation regressed in EAE after treatment with T-(B7H1). Sections of spinal cord
of mice sacrificed on day 15?20 postsensitization were stained with Luxol-fast blue (D) or H&E (E). Photomicrographs show the following: 1) normal
appearing dorsal columns in healthy mice; 2) severe demyelination in unstained areas in dorsal columns and the intensive mononuclear infiltration in spinal
cord in EAE mice treated with T-control; 3) marked inhibition of demyelination in dorsal columns and spinal cords, resulting in reduced inflammation, in
EAE mice receiving T-(B7H1) cells. Examples are representative of three independent experiments.
Down-regulatory effect of T-(B7H1) as Tr1 on peptide MOG35–55-induced EAE. A, Suppressive effect of T-(B7H1) on the response of
the percentages of Foxp3?and Nrp1?cells remained unchanged after treatment with B7H1-Ig. There was little expression of Foxp3 and Nrp1 for
B7H1-Ig-costimulated CD4?CD25?T cells. B, The expression intensity of CD45RB changed from high (Y-mean 1236) to low (Y-mean 462) in the CD4?
T cells twice stimulated with B7H1-Ig. C, Similar suppressive effects of T-(B7H1) and CD4?CD25?T-(B7H1) on MLC (syngeneic naive CD4?T cells ?
allogeneic splenocytes). T-(control) stands for CD4?T cells stimulated twice with anti-CD3 plus Ig-control. D, Effects of treatment with anti-IFN-?R and
anti-IL-10R mAbs on prevention of CD4?T cell development to Tr1. The T cells costimulated twice with B7H1-Ig plus either anti-IFN-?R, anti-IL-10R,
or Ig-control, referred to as T-(B7H1 ? anti-IFN-?R), T-(B7H1 ? anti-IL-10R), and T-(B7H1 ? Ig-control), respectively. Both T-(B7H1 ? anti-IFN-?R) and T-(B7H1 ?
anti-IL-10R), as well as the T-(B7H1) from the IFN-??/?mice failed to induce suppression of CD4?T cell response to allogeneic splenocytes when cocultured
in MLC. WT, wild-type C57BL/6; IFN-??/?, IFN-? gene-knockout C57BL/6.
CD4?T cells costimulated twice with B7H1-Ig are phenotypically and functionally equal to Tr1. A, For total T cells and the indicated subsets,
3612 B7H1-INDUCED Tr1 DIFFERENTIATION DEPENDS ON IFN-?
demonstrates that B7H1-Ig fusion protein, along with anti-CD3
mAb, can stimulate the generation of Tr1 from CD4?T cells.
In our testing system, naive CD4?CD62L?T cells (purity
?97%) were stimulated with anti-CD3 mAb and fusion protein
B7H1-Ig. This excludes possible contamination with accessory cells
or APCs, especially those capable of secreting IL-12 for Th1 dif-
ferentiation. Additional examination using ELISA indicated that
our naive T cells were negative for IL-12 production (data not
shown). This strongly implies that some DC-independent path-
ways for Tr1 differentiation exist.
Because IL-10 is essential for Tr1 activation and development,
autocrine mechanism might be used by the cells via their IL-10R.
This study not only confirms the role of IL-10 and its receptor, but
also reports for the first time a dependence upon IFN-? for Tr1
differentiation. In our APC-free system, IFN-? can be produced
only from CD4?T cells. It is apparent that the IFN-?-secreting T
cells are typical Th1 cells with a CD4?CCR5?CxCR3?T-
bet?Gata3?phenotype and a cytokine-secreting profile of IFN-
??IL-2?IL-4?IL-10?as identified in our experiments (Figs. 2, 4,
and 6). As reported, Tr1 cells often develop alongside Th1 cells in
several kinds of diseases, including autoimmune diseases, cancer,
infectious disease, and allergies (21). It is possible that when Th1
cells exert their effects by production of IFN-?, the generation of
Tr1 cells follows. This creates a feedback mechanism down-reg-
ulating the elevated immune responses initiated by Th1 cells.
In contrast, when CD4?T cells were stimulated with anti-CD3
plus anti-CD28, there appeared a typical Th1-related reaction pat-
tern with no evidence for generation of Tr1 (Fig. 1). The critical
role of costimulatory molecule B7H1 is therefore more impressive
for Tr1 differentiation, confirming our previous observation that
B7H1-expressing keratinocytes were active in inducing Tr1 differ-
entiation (42). In this sense, two kinds of receptors binding the
B7H1 molecule draw our attention. One is the ITIM-bearing PD-1
receptor (44, 45), which usually delivers an inhibitory signal to
stop the activation pathway initiated by the ITAM-bearing recep-
tors on the same cells. In our experiment, PD-1 was detected on T
cells, and its suppressive role in the late phase was confirmed by
blocking with mAb (Fig. 3A). The other receptor, however, is un-
known, although its importance has been suggested for induction
of stimulation or up-regulation instead of suppression (45). It is
possible that, in our experiments, B7H1 acted via such a receptor to
induce the IFN-?-secreting Th1 cells in the early phase as eluci-
dated by a hypothetic diagram we proposed (Fig. 9). The anti-
CD3/B7H1-Ig-activated Th1, in addition to producing large
amounts of IFN-?, might deliver some special signal(s) for Tr1
differentiation or via B7H1-engaged PD-1. It is evident that this
signal could not be produced by the anti-CD28-costimulated Th1
cells (Figs. 1A and 4). In this manner, the function of PD-1 would
not be restricted to delivery inhibitory signals on the late phase, but
also to induce the differentiation of Tr1 cells. The underlying
mechanisms need further investigation.
Transcription factor Foxp3 has been regarded critical for differ-
entiation of CD4?CD25?Treg and used as Treg unique marker to
distinguish from effector CD4?CD25?T cells (25, 26). In con-
trast, there are reports that Foxp3 was also detectable for
CD4?CD25?T cells stimulated with anti-CD3/anti-CD28 (56) or
TGF-? (57). However, there are no reports dealing with Foxp3-
positive Tr1 cells generated by stimulation with IL-10. This sug-
gests that whether Foxp3 is involved in activation of CD4?CD25?
Treg might depend on different stimuli and/or inducing systems.
Fig. 7A indicates that, however, the CD4?CD25?IFN-?R?IL-
10R?Tr1 cells we identified are Foxp3 negative.
The authors have no financial conflict of interest.
1. Sakaguchi, S. 2004. Naturally arising CD4?regulatory T cells for immunological
self-tolerance and negative control of immune responses. Annu. Rev. Immunol.
2. Shevach, E. M. 2002. CD4?CD25?suppressor T cells: more questions than
answers. Nat. Rev. Immunol. 2: 389–400.
3. Bluestone, J. A., and A. K. Abbas. 2003. Natural versus adaptive regulatory T
cells. Nat. Rev. Immunol. 3: 253–257.
4. Levings, M. K., and M. G. Roncarolo. 2005. Phenotypic and functional differ-
ences between human CD4?CD25?and type 1 regulatory T cells. Curr. Top.
Microbiol. Immunol. 293: 303–326.
5. Foussat, A., F. Cottrez, V. Brun, N. Fournier, J. P. Breittmayer, and H. Groux.
2003. A comparative study between T regulatory type 1 and CD4?CD25?T cells
in the control of inflammation. J. Immunol. 171: 5018–5026.
6. Von Herrath, M. G., and L. C. Harrison. 2003. Antigen-induced regulatory T cells
in autoimmunity. Nat. Rev. Immunol. 3: 223–232.
7. Wraith, D. C., K. S. Nicolson, and N. T. Whitley. 2004. Regulatory CD4?T cells
and the control of autoimmune disease. Curr. Opin. Immunol. 16: 695–701.
8. Segal, B. M., D. D. Glass, and E. M. Shevach. 2002. Cutting edge: IL-10-pro-
ducing CD4?T cells mediate tumor rejection. J. Immunol. 168: 1–4.
9. Akasaki, Y., G. Liu, N. H. Chung, M. Ehtesham, K. L. Black, and J. S. Yu. 2004.
Induction of a CD4?T regulatory type 1 response by cyclooxygenase-2-over-
expressing glioma. J. Immunol. 173: 4352–4359.
10. Zhang, X., H. Huang, J. Yuan, D. Sun, W. S. Hou, J. Gordon, and J. Xiang. 2005.
CD4?8?dendritic cells prime CD4?T regulatory 1 cells to suppress antitumor
immunity. J. Immunol. 175: 2931–2937.
11. Chattopadhyay, S., N. G. Chakraborty, and B. Mukherji. 2005. Regulatory T cells
and tumor immunity. Cancer Immunol. Immunother. 54: 1153–1161.
12. Wood, K. J., and S. Sakaguchi. 2003. Regulatory T cells in transplantation tol-
erance. Nat. Rev. Immunol. 3: 199–210.
13. Albert, M. H., Y. Liu, C. Anasetti, and X. Z. Yu. 2005. Antigen-dependent sup-
pression of alloresponses by Foxp3-induced regulatory T cells in transplantation.
Eur. J. Immunol. 35: 2598–2607.
14. Akdis, M., J. Verhagen, A. Taylor, F. Karamloo, C. Karagiannidis, R. Crameri,
S. Thunberg, G. Deniz, R. Valenta, H. Fiebig, et al. 2004. Immune responses in
healthy and allergic individuals are characterized by a fine balance between al-
lergen-specific T regulatory 1 and T helper 2 cells. J. Exp. Med. 199: 1567–1575.
15. Hawrylowicz, C. M., and A. O’Garra. Potential role of interleukin-10-secreting
regulatory T cells in allergy and asthma. 2005. Nat. Rev. Immunol. 5: 271–283.
16. Bellinghausen, I., B. Konig, I. Bottcher, J. Knop, and J. Saloga. 2005. Regulatory
activity of human CD4?CD25?T cells depends on allergen concentration, type
of allergen and atopy status of the donor. Immunology 116: 103–111.
CD4?T cells costimulated with B7H1-Ig and the interactions between Tr1
and Th1 cells. IFN-?-secreting CD4?Th1 cells are differentiated from the
naive CD4?T cells after stimulation with anti-CD3 mAb and costimula-
tory molecule B7H1. For activation of Th1, B7H1 uses an unidentified
activation receptor. Tr1 cells are further activated and maintained by liga-
tion of their IFN-?R and IL-10R with IFN-? and IL-10, respectively, in an
autocrine manner. It is also possible that for Tr1 activation some unknown
signals are delivered from the Th1 after the well-defined PD-1 is engaged
with B7H1 (PD-L1). High levels of IFN-?R and IL-10R production from
Tr1, together with the down-regulatory role exerted by the ITIM-bearing
PD-1 on Th1 upon ligated with B7H1, result in strong suppression and/or
some apoptosis for the CD4?Th1 cells, which are dominants for MLR and
autoimmune disease EAE. Right part of Fig. 9 indicates the differential
effects of blocking IFN-?, IL-10, TGF-?, and PD-1 with mAbs.
A hypothetic diagram for Tr1 differentiation from the
3613The Journal of Immunology
17. Akbani, P., G. J. Freeman, E. H. Meyer, E. A. Greenfield, T. T. Chang, Download full-text
A. H. Sharpe, G. Berry, R. H. DeKruyff, and D. T. Umtsu. 2002. Antigen specific
regulatory T cells develop via the ICOS-ICOS ligand pathway and inhibit aller-
gen-induced airway hyperreactivity. Nat. Med. 8: 1024–1032.
18. Satoguina, J., M. Mempel, J. Larbi, M. Badusche, C. Loliger, O. Adjei,
G. Gachelin, B. Fleischer, and A. Hoerauf. 2002. Antigen-specific T regulatory-1
cells are associated with immunosuppression in a chronic helminth infection (on-
chocerciasis). Microbes Infect. 4: 1291–1300.
19. Foussat, A., F. Cottrez, V. Brun, N. Fournier, J. P. Breittmayer, and H. Groux.
2003. A comparative study between T regulatory type 1 and CD4?CD25?T cells
in the control of inflammation. J. Immunol. 171: 5018–5026.
20. Walther, M., J. E. Tongren, L. Andrews, D. Korbel, E. King, H. Fletcher,
R. F. Anderson, P. Bejon, F. Thompson, S. J. Dunachie, et al. 2005. Up-regula-
tion of TGF-?, FOXP3, and CD4?CD25?regulatory T cells correlates with more
rapid parasite growth in human malaria infection. Immunity 23: 287–296.
21. Mills, K. H., and P. McGuirk. 2004. Antigen-specific regulatory T cells: their
induction and role in infection. Semin. Immunol. 16: 107–117.
22. Belkaid, Y., and B. T. Rouse. 2005. Natural regulatory T cells in infection dis-
ease. Nat. Immunol. 6: 353–360.
23. Battaglia, M., C. Gianfrani, S. Gregori, and M. G. Roncarolo. 2004. IL-10-pro-
ducing T regulatory type 1 cells and oral tolerance. Ann. NY Acad. Sci. 1029:
24. Aluvihare, V. R., M. Kallinkourdis, and G. B. Alexander. 2004. Regulatory T
cells mediate tolerance to the fetus. Nat. Immunol. 5: 266–271.
25. Hori, S., T. Nomura., and S. Sakaguchi. 2003. Control of regulatory T cell de-
velopment by the transcription factor Foxp3. Science 299: 1057–1061.
26. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the
development and function of CD4?CD25?T regulatory cells. Nat. Immunol. 4:
27. Thompson, C., and F. Powrie. 2004. Regulatory T cells. Curr. Opin. Pharmacol.
28. O’Garra, A., P. L. Vieira, P. Vieira, and A. E. Goldfeld. 2004. IL-10 producing
and naturally occurring CD4?Tregs: limiting collateral damage. J. Clin. Invest.
29. Roncarolo, M. G., R. Bacchetta, C. Bordignon, S. Narula, and M. K. Levings.
2001. Type 1 T regulatory cells. Immunol. Rev. 182: 68–79.
30. Vieira, P. L., J. R. Christensen, S. Minaee, E. J. O’Neill, F. J. Barrat, A. Boonstra,
T. Barthlott, B. Stockinger, D. C. Wraith, and A. O’Garra. 2004. IL-10-secreting
regulatory T cells do not express Foxp3 but have comparable regulatory function
to naturally occurring CD4?CD25?regulatory T cells. J. Immunol. 172:
31. Groux, H. 2003. Type 1 T-regulatory cells: their role in the control of immune
responses. Transplantation 75: 8S–12S.
32. Wakkach, A., N. Fournier, V. Brun, J. P. Breittmayer, F. Cottrez, and H. Groux.
2003. Characterization of dendritic cells that induce tolerance and T regulatory 1
cell differentiation in vivo. Immunity 18: 605–617.
33. Lundqvist, A., A. Palmborg, M. Pavlenko, J. Levitskaya, and P. Pisa. 2005.
Mature dendritic cells induce tumor-specific type 1 regulatory T cells. J. Immu-
nother. 28: 229–235.
34. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, and A. H. Enk. 2000. Induction of
interleukin 10-producing, nonproliferating CD4?T cells with regulatory prop-
erties by repetitive stimulation with allogeneic immature human dendritic cells.
J. Exp. Med. 192: 1213–1222.
35. Leving, M. K., S. Gregori, E. Tresoldi, S. Cazzaniga, C. Bonini, and
M. G. Roncarolo. 2005. Differentiation of Tr1 cells by immature dendritic cells
requires IL-10 but not CD25?CD4?Tr cells. Blood 105: 1162–1169.
36. Bacchetta, R., C. Sartirana, M. K. Levings, C. Bordignon, S. Narula, and
M. G. Roncarolo. 2002. Growth and expansion of human T regulatory type 1 cells
are independent from TCR activation but require exogenous cytokines. Eur.
J. Immunol. 32: 2237–2245.
37. Levings, M. K., R. Sangregorio, F. Galbiati, S. Squadrone, M. R. de Waal, and
M. G. Roncarolo. 2001. IFN-? and IL-10 induce the differentiation of human type
1 T regulatory cells. J. Immunol. 166: 5530–5539.
38. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul,
R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, and A. O’Garra. 2002.
In vitro generation of interleukin 10-producing regulatory CD4?T cells is in-
duced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and
Th2-inducing cytokines. J. Exp. Med. 195: 603–616.
39. O’Neill, E. J., E. Sawicka, C. Manlius, M. A. Le, L. R. Brunet, D. M. Kemeny,
G. Bowen, G. Rook, and C. Walker. 2004. Natural and induced regulatory T cells.
Ann. NY Acad. Sci. 1029: 180–192.
40. Yang, C. C., T. S. Shih, T. A. Chuh, W. S. Hsu, S. Y. Kuo, and Y. F. Chao. 1980.
The intermingled transplantation of auto- and homografts in severe burns. Burns
41. Yang, C. C., T. S. Shih, and W. S. Wu. 1982. A Chinese concept of treatment of
extensive third-degree burns. Plast. Reconstr. Surg. 70: 238–242.
42. Cao, Y., H. Zhou, J. Tao, Z. Zheng, N. Li, B. Shen, T. S. Shih, J. Hong, J. Zhang,
and K. Y. Chou. 2003. Keratinocytes induce local tolerance to skin graft by
activating interleukin-10-secreting T cells in the context of costimulation mole-
cule B7–H1. Transplantation 75: 1390–1396.
43. Dong, H., G. Zhu, K. Tamada, and L. Chen. 1999. B7–H1, a third member of the
B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat.
Med. 5: 1365–1369.
44. Freeman, G. J., A. J. Long, Y. Iwai, K. Bourque, T. Chernova, H. Nishimura,
L. J. Fitz, N. Malenkovich, T. Okazaki, M. C. Byrne, et al. 2000. Engagement of
the PD-1 immunoinhibitory receptor by a novel B7 family member leads to
negative regulation of lymphocyte activation. J. Exp. Med. 192: 1027–1034.
45. Tamura, H., H. Dong, G. Zhu, G. L. Sica, D. B. Flies, K. Tamada, and L. Chen.
2001. B7–H1 costimulation preferentially enhances CD28-independent T-helper
cell function. Blood 97: 1809–1816.
46. Oliver, A. R., G. M. Lyon, and N. H. Ruddle. 2003. Rat and human myelin
oligodendrocyte glycoproteins induce experimental autoimmune encephalomy-
elitis by different mechanisms in C57BL/6 mice. J. Immunol. 171: 462–468.
47. Tompkins, S. M., J. Padilla, M. C. Dal Canto, J. P. Ting, K. L. Van, and
S. D. Miller. 2002. De novo central nervous system processing of myelin antigen
is required for the initiation of experimental autoimmune encephalomyelitis.
J. Immunol. 168: 4173–4183.
48. Kohm, A. P., P. A. Carpentier, H. A. Anger, and S. D. Miller. 2002. Cutting edge:
CD4?CD25?regulatory T cells suppress antigen-specific autoreactive immune
responses and central nervous system inflammation during active experimental
autoimmune encephalomyelitis. J. Immunol. 169: 4712–4716.
49. Bruder, D., M. Probst-Kepper, A. M. Westendorf, R. Geffers, S. Beissert,
K. Loser, H. von Boehmer, J. Buer, and W. Hansen. 2004. Neuropilin-1: a surface
marker of regulatory T cells. Eur. J. Immunol. 34: 623–630.
50. Loetscher, P., M. Uguccioni, L. Bordoli, M. Baggiolini, B. Moser, C. Chizzolini,
and J. M. Dayer. 1998. CCR5 is characteristic of Th1 lymphocytes. Nature 391:
51. Murphy, K. M., and S. L. Reiner. 2002. The lineage decisions of helper T cells.
Nat. Rev. Immunol. 2: 933–944.
52. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression of
the eotaxin receptor CCR3 by human T helper 2 cells. Science 277: 2005–2007.
53. Chen, L. 2004. Co-inhibitory molecules of the B7-CD28 family in the control of
T-cell immunity. Nat. Rev. Immunol. 4: 336–347.
54. Foussat, A., F. Cottrez, V. Brun, N. Fournier, J. P. Breittmayer, and H. Groux.
2003. A comparative study between T regulatory type 1 and CD4?CD25?T cells
in the control of inflammation. J. Immunol. 171: 5018–5026.
55. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, and F. Powrie. 1999. An
essential role for interleukin 10 in the function of regulatory T cells that inhibit
intestinal inflammation. J. Exp. Med. 190: 995–1004.
56. Walker, M. R., D. J. Kasprowicz, V. H. Gersuk, A. Benard, M. Van Landeghen,
J. H. Buckner, and S. F. Ziegler. 2003. Induction of FoxP3 and acquisition of T
regulatory activity by stimulated human CD4?CD25?T cells. J. Clin. Invest.
57. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, and
S. M. Wahl. 2003. Conversion of peripheral CD4?CD25?naive T cells to
CD4?CD25?regulatory T cells by TGF-? induction of transcription factor
Foxp3. J. Exp. Med. 198: 1875–1886.
3614B7H1-INDUCED Tr1 DIFFERENTIATION DEPENDS ON IFN-?