of June 13, 2013.
This information is current as
Regulatory T Cells
through IL-10: Novel Suppressive Mode for
Cutting Edge: Induction of B7-H4 on APCs
Mottram, Huanbin Xu, Lieping Chen and Weiping Zou
Ilona Kryczek, Shuang Wei, Linhua Zou, Gefeng Zhu, Peter
2006; 177:40-44; ;
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cites 19 articles
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2006 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
Cutting Edge: Induction of B7-H4 on APCs through IL-10:
Novel Suppressive Mode for Regulatory T Cells1
Ilona Kryczek,* Shuang Wei,* Linhua Zou,* Gefeng Zhu,†Peter Mottram,‡Huanbin Xu,‡
Lieping Chen,†and Weiping Zou2*
Multiple modes of suppressive mechanisms including
IL-10 are thought to be implicated in CD4?CD25?reg-
ulatory T (Treg) cell-mediated suppression. However, the
cellular source, role, and molecular mechanism of IL-10
the interaction between Treg cells and APCs. We demon-
strate that Treg cells, but not conventional T cells, trigger
high levels of IL-10 production by APCs, stimulate APC
B7-H4 expression, and render APCs immunosuppressive.
Initial blockade of B7-H4 reduces the suppressive activity
mediated by Treg cell-conditioned APCs. Further, APC-
derived, rather than Treg cell-derived, IL-10 is responsi-
ble for APC B7-H4 induction. Therefore, Treg cells con-
vey suppressive activity to APCs by stimulating B7-H4
expression through IL-10. Altogether, our data provide a
novel cellular and molecular mechanism for Treg cell-me-
diated immunosuppression at the level of APCs, and sug-
gest a plausible mechanism for the suppressive effect of
IL-10 in Treg cell-mediated suppression. The Journal of
Immunology, 2006, 177: 40–44.
modes are proposed to explain the suppressive mechanisms of
regulatory T (Treg)3cells (5). Some in vitro experimental data
nonredundant role of IL-10 in Treg cell-mediated suppression
(8–10). Nonetheless, the source and molecular suppressive
B7-H4 is a recently discovered B7 family member. B7-H4
negatively regulates T cell responses in vitro (11–13). Treg cells
(2) and suppressive B7-H4?macrophages (14) were localized
in ovarian tumor. In this report, we studied the interaction be-
tween Treg cells and APCs. Our study reveals a previously un-
appreciated mechanistic relationship among IL-10, B7-H4,
reg cells comprise 5–10% of the circulating CD4?T
cell population and play a crucial role in different
pathological settings (1–4). Multiple suppressive
Treg cells, and APCs and demonstrates a novel molecular and
cellular suppressive mechanism for Treg cell-mediated immu-
Materials and Methods
(Treg cells), CD4?CD45RO?CD25?T cells, and lin?HLA-DR?CD11c?
Primary myeloid dendritic cells (MDCs) were sorted with FACSaria (BD Bio-
sciences) with purity ?98% (15). Monocyte-derived DCs (MDDCs) were ob-
tained as described (16). Cells were stained with mAbs and analyzed on a LSR
II (BD Biosciences). Mouse anti-human Abs, including CD4-FITC (SK3),
CD25-PE (MA251), HLA-DR-PercP (L243), CD11c-allophycocyanin (B-
ly6), and CD14-allophycocyanin-Cy7 (M?P9) were obtained from BD Bio-
Human FOXP3 detection
RT-PCR was conducted for FOXP3 (2). Results were expressed as fold differ-
ences relative to GAPDH (2). FOXP3 protein was detected by intracellular
staining with rat anti-human FOXP3 Ab (clone PCH101; eBioscience).
Human T cell immunosuppressive assay
T cell immunosuppression was tested in a coculture system. CD4?CD25?T
cells (2 ? 105/ml) were stimulated with 2.5 ?g/ml anti-human CD3 (clone
UCHT1), 1.2 ?g/ml anti-human CD28 (clone CD28.2) (BD Biosciences),
Treg cells or different concentrations of conditioned CD14?cells as indicated.
Seventy-two hours after coculture, T cell proliferation was evaluated by thymi-
dine incorporation. In some cases, CD14?cells were incubated with mouse
anti-human IL-10 receptor (0.5 ?g/ml, mouse IgG1, clone 37607; R&D Sys-
tems) as indicated.
C57BL wild-type and IL-10?/?mice (The Jackson Laboratory) were main-
tained in specific pathogen-free conditions. Eight-week-old female mice were
used in all the experiments. Mouse CD11b?cells, CD4?CD25?cells, and
CD4?CD25?T cells were enriched from spleen cells with mouse CD4 selec-
tion kits (StemCell Technology) and sorted with high purity (? 95%). Mouse
T cell immunosuppression was tested in a coculture system. CD4?CD25?T
2C11; BD Biosciences) and fresh CD11b?cells (2 ? 105/ml) in the presence
of different concentrations of CD4?CD25brightT cells (Treg cells). Seventy-
two hours after coculture, T cell proliferation was evaluated by thymidine in-
*Department of Surgery, University of Michigan, Ann Arbor, MI 48109;†Department of
Dermatology, Johns Hopkins University, Baltimore, MD 21287; and‡Tulane University
Health Sciences Center, New Orleans, LA 70112
Received for publication November 8, 2005. Accepted for publication May 3, 2006.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1This work was supported by National Cancer Institute Grants CA092562, CA100227,
and CA99985 (to W.Z.) and CA97085 (to L.C.).
2Address correspondence and reprint requests to Dr. Weiping Zou, Department of Sur-
gery, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346. E-mail address:
3Abbreviations used in this paper: Treg, regulatory T; MDC, primary myeloid dendritic
cell; MDDC, monocyte-derived DC.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
by guest on June 13, 2013
Expression and regulation of human APC B7-H4 expression
with human rIL-10 (R&D Systems) or with different concentrations of autol-
ogous CD4?CD25?T cells, Treg cells (0–1 ? 106/ml) in the presence of
anti-human CD3, and anti-human CD28. In some cases, cytokines were de-
against human IL-10 (clone 23738, 50 ng/ml; R&D Systems) was used as in-
dicated. B7-H4 mRNA was detected by RT-PCR (17). To detect B7-H4 pro-
tein, cells were initially incubated with human AB serum to block nonspecific
controls were cells stained with medium, primary mAb, second mAb with or
without isotypes. B7-H4 surface protein was analyzed by flow cytometry.
Blockade of human B7-H4 induction
Antisense morpholino oligonucleotide specific for B7-H4 (GAGGATCTGC
CCCAGGGAAGCCATG) (B7-H4 blocking oligos), and the inverted oligo-
nucleotide (control oligos) were produced by GeneTools. To block B7-H4 in-
duction, monocytes were incubated for 3 h with 0.6 ?M oligos in serum-free
medium supplemented with 0.2 ?M ethoxylated polyethylenimine (Gene-
Tools). Cells were washed twice and used for additional experiments.
Differences in cell surface molecule expression were determined by ?2test, and
in other variables by unpaired t test, with p ? 0.05 considered significant.
Treg cells, but not conventional T cells, trigger APC-dependent, high
levels of IL-10
expressed FOXP3 mRNA (n ? 12; ?, p ? 0.001) (Fig. 1A).
population (17). FOXP3?T cells were largely found in
CD4?CD25brightT cell population (Fig. 1B). CD4?CD25bright
T cells inhibited T cell proliferation in a dose-dependent manner
(n ? 10; ?, p ? 0.01, ??p ? 0.0001) (Fig. 1C). Therefore, we
sorted CD4?CD25brightT cells (Treg cells) for our experiments.
We studied the interaction between APCs and Treg cells. Treg
cells or CD4?CD25?T cells or CD4?CD45RO?CD25?T
cells were cocultured for 72 h with autologous monocytes in the
els of IL-10 in the culture supernatants with Treg cells than con-
TNF-?, but not IL-10, was higher in the culture with
CD4?CD45RO?CD25?T cells (632 ? 87 pg/ml) than with
pg/ml) (n ? 4; p ? 0.01), whereas there was no significant differ-
gest that Treg cells, but not conventional T cells, may selectively
trigger high levels of IL-10 production.
We detected little IL-10 in the culture supernatants when
monocytes were omitted. Addition of monocytes resulted in
significantly higher levels of IL-10 production in Treg cell and
monocyte coculture (n ? 7; ?, p ? 0.001) (Fig. 1D). Similar
and MDDCs. Altogether, our data demonstrate that Treg cells
condition APC-dependent, high levels of IL-10 production.
Treg cell suppressive capacity is reduced in the absence of IL-10 in APCs
To determine the importance of the high levels of IL-10 trig-
gered by Treg cells, we sorted CD11b?monocytes, Treg cells,
and CD4?CD25?T cells from wild-type and IL-10?/?mice.
We cocultured autologous Treg cells with T cells and CD11b?
cells in the presence of anti-CD3 mAb. As expected, IL-10?/?
and IL-10?/?Treg cells exhibited a dose-dependent suppres-
sive activity in vitro. However, the levels of suppression were
significantly higher in the group with all the cells from IL-
10?/?mice than from IL-10?/?mice (n ? 5; p ? 0.05) (Fig.
2A). Thus, although IL-10 is not essential for Treg cell
Treg cell-mediated suppression.
To determine which population is responsible for IL-10 pro-
duction, we used various combinations of cells from IL-10?/?
and IL-10?/?mice (Fig. 2B). We showed that when the ratio
between Treg cells and CD4?CD25?T cells was ?1:1, the
addition of IL-10?/?CD11b?cells resulted in significant re-
duction of Treg cell-mediated suppression (n ? 5; ?, p ? 0.05,
compared with IL-10?/?CD11b?cells) (Fig. 2B and data not
shown). When the ratio between Treg cells (2 ? 105/ml) and
CD4?CD25?T cells (2 ? 105/ml) was 1:1, the levels of Treg-
among different cellular combinations. It suggests that APC-
derived IL-10 is critical for Treg cell suppressive activity.
affected Treg cell-mediated suppression (p ? 0.05) (Fig. 2B).
Our data suggest that APC-dependent, but not Treg cell-de-
pendent IL-10, implicates in Treg cell-mediated suppression.
Treg cells enable APC suppressive activity
Our human studies suggest that Treg cells triggered APC-de-
10?/?mice indicated that APC-derived IL-10 implicates in
A, CD4?CD25?T cells express FOXP3 mRNA. CD4?CD25brightT cells
were sorted by FACS from blood. FOXP3 mRNA was determined by RT-PCR
as described (2, 20). Inset, FOXP3 RT-PCR products migrated in the gel.;
CD25?, Treg cells; CD25?, CD4?CD25?T cells; Control, no cDNA input.
by intracellular staining (gated on CD4?CD3?cells) (see Materials and Meth-
ods). Results were expressed as the percent of FOXP3?in CD4?T cells (upper
cells (histogram, lower panel). n ? 7. C, Treg cells suppress T cell activation in
vitro. CD3?CD25?T cells were cultured with different concentrations of au-
tologous Treg cells as indicated. T cell proliferation was determined by thymi-
dine incorporation. Results are expressed as the mean cpm ? SEM. D, Treg
cells induce APC-dependent IL-10 production. Autologous Treg cells and
conventional T cells were cocultured in the presence or absence of CD14?
cells as described. The culture supernatants were collected, and IL-10 was
detected in the supernatants with ELISA kit. Results are expressed as the
mean value ? SEM.
41The Journal of Immunology
by guest on June 13, 2013
Treg cells suppressive activity (Fig. 2). We asked whether Treg
cell-conditioned APCs are distinct from conventional T cell-
conditioned APCs. To this end, we incubated CD14?cells
with Treg cells or conventional T cells in the presence of anti-
human CD3 and anti-human CD28. Seventy-two hours later,
we sorted these monocytes and tested their capacity of activat-
ing T cells. Strikingly, CD14?cells pretreated with Treg cells,
but not CD4?CD25?T cells and medium, significantly sup-
pressed T cell proliferation (n ? 5; ? p ? 0.05) (Fig. 3A). The
data indicate that Treg cells enable APC suppressive activity.
To study the role of IL-10 in T cell suppression mediated by
Treg cell-conditioned APCs, Treg cell-conditioned CD14?
cells were preincubated with mouse anti-human IL-10 recep-
presence of anti-IL-10 receptor. We observed that T cell sup-
pression mediated by Treg cell-conditioned CD14?cells was
mediated by Treg cell-conditioned APCs.
Treg cells selectively stimulate APC B7-H4 expression
We further studied the phenotype of CD14?cells conditioned
by Treg cells and conventional T cells. Treg cells, but not con-
ventional T cells, including CD4?CD45RO?CD25?T cells,
stimulated B7-H4 expression on CD14?cells (n ? 12; ?, p ?
0.001) (Fig. 3, C–E, and data not shown). We observed similar
expression of CD40, CD54, CD80, and HLA class I and II ex-
ditioned monocytes. It suggests that Treg cells may selectively
stimulate B7-H4 expression on monocytes.
To determine whether Treg cells stimulate B7-H4 in differ-
ent APC subsets, we cocultured Treg cells with MDCs or MD-
DCs. We showed that Treg cells significantly stimulated
B7-H4 expression on MDCs and MDDCs (n ? 8; ?, p ? 0.01
for all) (Fig. 3, D and E). The data indicate that Treg cells can
induce B7-H4 expression on multiple APC subsets.
Induction of APC B7-H4 is IL-10 dependent
Treg cells trigger high levels of IL-10 production (? 1 ng/ml)
(Fig. 1D). We hypothesized that IL-10 may contribute to
B7-H4 induction on APCs. To test this, we analyzed B7-H4
expression on CD14?cells during coculture with Treg cells in
the presence of neutralizing Ab against IL-10. Anti-human
IL-10 partially but significantly decreased CD14?cell B7-H4
expression (n ? 6; ?, p ? 0.001) (Fig. 4A). In support of this,
rIL-10 stimulated B7-H4 expression on APCs (n ? 6; p ?
0.001) (Fig. 4, B and C). Further, low concentrations of IL-10
IL-10 in APCs. Mouse Treg suppressive capacity is reduced in the presence of
IL-10?/?APCs. Treg cells, CD4?CD25?T cells and CD11b?cells were
sorted from wild-type and IL-10?/?mice. Variable concentration of Treg cells
were cocultured with 2 ? 105/ml CD4?CD25?T cells and CD11b?cells
proliferation was determined by thymidine incorporation. A, Treg-mediated
suppression in IL-10?/?vs IL-10?/?mice. B, Treg-mediated suppression in
different combinations of cells from IL-10?/?and IL-10?/?mice. Treg-me-
diated suppression was analyzed with different combinations of cells sorted
of suppression was calculated with the formula: 100% ? 100 ? cpm values
cells were used. Results were shown at 4 ? 104/ml Treg cells. IL-10?/?, cells
from IL-10?/?mice; W, cells from wild-type mice.
Mouse Treg suppressive capacity is reduced in the absence of
Treg cell-conditioned monocytes are suppressive. Fresh CD14?cells were cul-
tured with autologous Treg cells or CD4?CD25?T cells or medium in the
sorted from this coculture. Different concentrations of the sorted CD14?cells
of anti-CD3 and anti-CD28 mAbs. T cell proliferation was detected by thymi-
dine incorporation. Results are expressed as the mean cpm ? SEM. B, IL-10 is
above culture system, CD14?cells were initially incubated with anti-human
ml) were subject to T cell activation in the presence of anti-human IL-10 re-
ceptor. Results are expressed as the mean percentage of T cell proliferation ?
SEM. C–E, Treg cells stimulate B7-H4 expression on APCs. APC subsets were
cocultured with different concentrations of Treg cells or CD4?CD25?T cells
for 3 days in the presence of anti-CD3 and anti-CD28 mAbs. B7-H4 expres-
sion was analyzed by FACS. B7-H4 expression was expressed as the mean per-
centage of positive cells ? SEM in gated CD14?cells (C and D) and CD11c?
cells (D). B7-H4 expression also was expressed by histogram (E). Treg cell con-
centration was 1 ? 106/ml for D and E.
Human Treg cells render CD14?cells immunosuppressive. A,
Induction of APC B7-H4 by Treg cells is IL-10 dependent. Autologous Treg
cells were cultured with CD14?cells with anti-CD3 and anti-CD28 mAbs for
3 days in the presence of anti-human IL-10 or and control mAb. B7-H4 ex-
APC subsets. APC subsets were cultured for 72 h in medium or different con-
the mean percentage of positive cells ? SEM (A and B) or histogram (C).
Induction of APC B7-H4 by Treg cells is IL-10 dependent. A,
42CUTTING EDGE: APC B7-H4 AND Treg CELLS
by guest on June 13, 2013
(0.1–1 ng/ml) efficiently stimulated B7-H4 expression on
APCs (Fig. 4B), but not CD40, CD80, and CD86 expression
(data not shown). Thus, Treg cells trigger APC B7-H4 expres-
sion at least partially through IL-10.
Blocking B7-H4 reduces the suppressive activity of Treg cell-conditioned
We next determined whether APC B7-H4 is involved in the
Treg cell-suppressive capacity. Specific neutralizing anti-
human B7-H4 mAb is not available. We designed the B7-H4
blocking oligos and control oligos.
We initially studied IL-10-stimulated normal monocytes.
The B7-H4 blocking oligos, but not control oligos, signifi-
cantly inhibited basal and IL-10-induce B7-H4 mRNA expres-
blocked B7-H4 protein induced by IL-10 (n ? 6; p ? 0.05)
(Fig. 5B). Neither B7-H4 blocking oligos nor control oligos af-
fected macrophage MHC class I, MHC class II, CD16, CD32,
CD80, or CD86 expression (data not shown).
We next examined whether B7-H4 blocking oligo would
oligos or control oligos and then cocultured with Treg cells.
The B7-H4 blocking, but not control oligos, significantly re-
duced monocyte B7-H4 expression induced by Treg cells,
whereas control oligos had no significant effects on B7-H4 ex-
B7-H4 blocking oligos specifically block Treg-induced mono-
cyte B7-H4 expression.
We further evaluated the role of B7-H4 in T cell suppression
3A). To this end, we initially exposed normal monocytes to
B7-H4 blocking oligos or control oligos or medium. These
monocytes were subsequently incubated with Treg cells for
72 h. We then sorted these monocytes for testing their capacity
of activating T cells. As expected, T cell suppression was signif-
exposed to B7-H4 blocking oligos, compared with control oli-
gos and medium (n ? 6; ?, p ? 0.01) (Fig. 5D). These data
indicate that B7-H4 contributes to the suppressive activity me-
diated by Treg cell-conditioned APCs.
In this study, we document that Treg cells trigger high levels of
IL-10 production by APCs and, in turn, stimulate APC B7-H4
expression in an autocrine manner and render APCs immuno-
suppressive via B7-H4.
Inconsistent with some reports (6, 18), we show that IL-
10?/?Treg cells are suppressive. Hence, Treg-derived IL-10 is
ical immunosuppressive assay (6), Treg suppressive capacity is
from IL-10?/?mice. It indicates that IL-10 participates in
Treg-mediated suppression. In support of this, when the ratio
between Treg cells and conventional T cells is ?1:1, the sup-
and profoundly reduced when IL-10?/?APCs are added. The
data indicate that APC, but not Treg-derived IL-10, is crucial
for Treg-mediated suppression.
of APCs, or APCs are substituted by irradiated whole spleen
cells. It is not a surprise that these assays cannot define the role
IL-10. APCs actively process Ags and present Ags to T cells in
reach 1:1 in vivo. Thus, APC-derived IL-10 would likely in-
volve in Treg-mediated T cell suppression in vivo.
Treg cells, but not conventional T cells, trigger high levels of
IL-10 has long been thought as an immunosuppressive cyto-
kine, but it remains elusive how IL-10 implicates in Treg-me-
by effects on APCs (7, 19). However, very high concentrations
(19). This concentration may not be physiologically relevant in
vivo. We now show that as low as 0.1 ng/ml IL-10 can pro-
APC IL-10 production, which in turn stimulates B7-H4 ex-
pression and renders APCs suppressive through B7-H4. Thus,
our data provide a plausible mechanism for the suppressive ef-
fect of IL-10. Because Treg cells are a small population, Treg-
to-T cell contact-dependent suppressive mechanism may not
cells may inhibit APC function. Induction of suppressive
B7-H4 on APCs provides a novel molecular and cellular basis
for Treg-mediated suppression in the level of APCs.
Our data mechanistically link IL-10, B7-H4, Treg cells, and
Although mouse B7-H4 ligation of T cells has a profound in-
hibitory effect T cell activation (11, 12), the regulatory mech-
anisms and function of B7-H4 remain to be defined. We show
dent. A, B7-H4 blocking oligos block monocyte B7-H4 mRNA. Blood mono-
cytes were exposed to B7-H4 blocking oligos or control oligos for 24 h with or
without IL-10. B7-H4 mRNA was detected by RT-PCR. n ? 9. B, B7-H4
blocking oligos block IL-10-induced monocyte B7-H4 protein. Blood mono-
cytes were exposed to B7-H4 blocking oligos and control oligos with IL-10 (10
ng/ml). Cell surface B7-H4 was detected by FACS analysis. Results were ex-
pressed as the mean percentage of positive cells ? SEM. C, B7-H4 blocking
tured with Treg cells as described. B7-H4 expression was detected by FACS by
gating on CD14?cells. Results were expressed as the mean percentage of pos-
itive cells ? SEM. D, Oligos exposed, Treg-conditioned CD14?cells were
sorted and further subject to stimulating autologous CD4?CD25?T cells as
sults were expressed as the mean cpm values ? SEM.
Supressive activity of Treg-conditioned APCs is B7-H4 depen-
43The Journal of Immunology
by guest on June 13, 2013