of June 4, 2013.
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and Specifically Suppress Effector T Cell
Accumulate in Kidney Allograft Tolerance
Myeloid-Derived Suppressor Cells
Martinet, Pamela Thebault, Karine Renaudin and Bernard
Vuillefroy de Silly, Claire Usal, Helga Smit, Bernard
Michèle Heslan, Fabienne Haspot, Nicolas Poirier, Romain
Anne-Sophie Dugast, Thomas Haudebourg, Flora Coulon,
2008; 180:7898-7906; ;
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The Journal of Immunology
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Myeloid-Derived Suppressor Cells Accumulate in Kidney
Allograft Tolerance and Specifically Suppress Effector T Cell
Anne-Sophie Dugast,* Thomas Haudebourg,* Flora Coulon,* Miche `le Heslan,*
Fabienne Haspot,* Nicolas Poirier,* Romain Vuillefroy de Silly,* Claire Usal,* Helga Smit,*
Bernard Martinet,* Pamela Thebault,* Karine Renaudin,†and Bernard Vanhove2*
The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe
unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3?class
II?CD11b?CD80/86?plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells
were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner.
Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated
allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly,
CD4?CD25highFoxP3?regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer
of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after
administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that
increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the
NO-dependent maintenance phase of tolerance. The Journal of Immunology, 2008, 180: 7898–7906.
ample, administration of anti-CD28 Abs (4) or anti-donor class II
Abs (5) results in tolerance to kidney allografts across a full MHC
barrier with an immune suppression that is at least partially de-
pendent on non-T cells. In most cases, non-T cell populations with
potential regulatory activity in the transplant setting have been
identified as regulatory dendritic cells producing IDO (6) and
heme oxygenase-1 (HO-1) (7), alternatively activated macro-
phages creating a Th2 environment (8), or NKT cells (9). Another
type of non-T regulatory cell, so-called myeloid suppressor cells,
have been associated with impaired immune reactivity to Ag chal-
lenge (mainly tumors but also infections), chronic inflammation, or
superantigen-induced tolerance of CD4?T lymphocytes (re-
lthough transplant tolerance in rats has frequently been
associated with the action of regulatory T cells (Tregs)3
(1–3), in some cases Tregs are not instrumental. For ex-
viewed by Sefarini et al. (10)). Myeloid suppressor cells have also
been found to contribute to the immunosuppression accompanying
the lethal systemic graft-vs-host reaction in irradiated mice (11).
These cells are a heterogeneous mixture of myeloid cells at dif-
ferent stages of differentiation, including precursors of granulo-
cytes, macrophages, dendritic cells, or early myeloid progenitors.
To avoid confusion between mesenchymal stem cells and the com-
monly used term “myeloid suppressor cells”, Gabrilovich et al.
proposed the term “myeloid-derived suppressor cells” (MDSC)
(12). In mice, these cells share the common functional character-
istics of being able to inhibit T cell responses by inducing the
apoptosis of activated T cells via up-regulation of NO production
(13). NO regulates T cell activation via reversible disruption of the
Jak/STAT5 signaling pathway (14). Additionally, arginase 1 can
be induced in MDSC under the action of Th2-type cytokines,
which synergize with NO to give rise to peroxynitrites that drive
the apoptosis of Ag-primed T lymphocytes by inhibiting protein
tyrosine phosphorylation via nitration of tyrosine residues (15). It
has also been suggested that MDSC suppressive activity is medi-
ated by CD4?CD25?regulatory T cells and requires an interaction
between CD152 on Tregs with CD80 that is up-regulated on
MDSC upon contact with activated T lymphocytes (16). Mouse
MDSC are defined phenotypically by their expression of CD11b
and Gr1 (Ly-6G). The human equivalents are less well defined;
they have been described as granulocyte-macrophage-progenitor
cells expressing the CD34 marker (17). The rat counterparts ex-
press the CD11b/c and HIS48 myeloid markers (18).
Having previously identified non-T cells with suppressive ac-
tivity in a rat model of kidney transplant tolerance (4), the aim of
the current study was to characterize the phenotype and mecha-
nisms of action of these cells. We report herein on an accumulation
of suppressor cells within the peripheral blood and grafts of tol-
erant kidney graft recipients. These cells are characterized by their
expression of CD80/86, signal regulatory protein ? (SIRP?,
*Institut National de la Sante ´ et de la Recherche Me ´dicale, U643, Centre Hospitalier
Universitaire Nantes, Institut de Transplantation et de Recherche en Transplantation,
Universite ´ de Nantes, Faculte ´ de Me ´decine, Nantes; and
Pathologique du Centre Hospitalier Universitaire de Nantes, Nantes, France
Received for publication November 13, 2007. Accepted for publication April
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 work was supported in part by the Roche Organ Transplant Research Foun-
dation Grant 466230972 (to B.V.) and by the Progreffe Fundation.
2Address correspondence and reprint requests to Dr. Bernard Vanhove, Institut de
Transplantation et de Recherche en Transplantation, Institut National de la Sante ´ et de
la Recherche Me ´dicale U643, Centre Hospitalier Universitaire Ho ˆtel Dieu, 30
boulevard Jean Monnet, 44093 Nantes Cedex 1, France. E-mail address:
3Abbreviations used in this paper: Treg, regulatory T cell; HO-1, heme oxygenase-1;
iNOS, inducible NO synthase; L-NMMA, NG-monomethyl-L-arginine; MDSC, my-
eloid-derived suppressor cells; 1-MT, 1-methyl-D,L-tryptophan; SIRP?, signal regu-
latory protein ?; SnPP, tin protoporphyrin.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
by guest on June 4, 2013
CD172a), CD11a/b, and inducible NO synthase (iNOS) and their
ability to suppress T cell proliferation in a contact-dependent and
iNOS-dependent manner. Additionally, our data demonstrate that
these MDSC selectively suppress activated effector T cells,
whereas natural CD4?CD25highTregs are largely resistant to this
Materials and Methods
Animals and transplantations
Eight- to 12-wk-old male Lewis.1W (LEW.1W, haplotype RT1u) and
Lewis.1A (LEW.1A, haplotype RT1a) congeneic rats (Centre d’Elevage
Janvier, Le Genest-Saint-Isle, France) differed in their entire MHC region.
Heterotopic LEW.1W kidney transplantation was performed as previously
described (19). The kidney (right side) of the recipient (LEW.1A) was
replaced by a LEW.1W donor allograft, and a contralateral nephrectomy
was performed 7 days later, after which the allograft was life-sustaining.
Studies described herein have been performed in accordance with the in-
stitutional guidelines of the Institut National de la Sante ´ et de la Recherche
Me ´dicale (INSERM).
The JJ319 (IgG1 anti-rat CD28) mouse hybridoma was a gift from Dr.
Thomas Hunig (Wurzburg, Germany). The JJ319 mAb was purified from
hybridoma supernatant and administered to LEW.1A allograft recipients by
i.p. injection at 1 mg/day for 7 days starting on the day of transplantation.
This Ab induces a transient down-modulation of CD28 expression in vivo,
without depleting target cells (20). Without treatment, the grafts were re-
jected 11 days posttransplantation. Syngeneic transplants (LEW.1A to
LEW.1A) served as controls.
Abs and reagents
Purified anti-B7-1 (clone 3H5) and anti-B7-2 (clone 24F) mouse hybrid-
omas were a gift from Dr. H. Yagita (Juntendo University School of Med-
icine, Tokyo, Japan). FITC-conjugated Pan-T (CD6), anti-CD11a
(WT.1), anti-CD11b (WT.5), anti-RT1A (MHC class I), anti-CD40, and
purified anti-CD45R (clone HIS24) were purchased from BD Pharmin-
gen. HIS48 Ab was from Serotec. FITC-conjugated anti-CD172a
(SIRP? or OX41), anti-CD4 (W3/25), anti-MHC class I (OX18), anti-
MHC class II (OX6), anti-CD8 (OX8), anti-CD25 (OX39), anti-CD103
(OX62), anti-CD90 (OX7), and anti-CD62L (OX85) were prepared in
our laboratory from the corresponding hybridomas obtained from the
European Cell Culture Collection (Salisbury, U.K.). Alexa Fluor 647-
conjugated anti-NKRP-1 (3.2.3) was prepared in our laboratory using a
conjugation kit (Invitrogen: Molecular Probes). PE-conjugated anti-
mouse was purchased from Jackson ImmunoResearch Laboratories. The
anti-TCR? R7-3 Ab was stained with a Zenon Alexa Fluor 700 mouse
IgG1 labeling kit from Molecular Probes according to the manufactur-
er’s instructions. Rabbit anti-rat iNOS Ab was purchased from Abcam.
Cells and cell sorting
Spleen T cells were prepared by nylon wool adhesion followed by deple-
tion of NK cells, B cells, and monocytes using specific mAbs (clones 3.2.3,
HIS24, and OX42, respectively), followed by anti-mouse IgG-coated
Dynabeads (Invitrogen). Blood sampling was performed in heparin tubes.
Erythrocytes were removed by hypotonic lysis. MDSC were identified by
staining 30 min at 4°C with anti-rat CD80/86 mAbs and PE-conjugated
anti-mouse IgG secondary Ab (Jackson ImmunoResearch Laboratories).
After saturation with an excess of mouse IgG, cells were stained with
FITC-conjugated anti-rat CD6 Ab and Alexa 647-conjugated anti-rat
NKRP-1 Ab. Cells were then filtered (60 ?m) and sorted using an Aria
flow cytometer (BD Biosciences). Cell purity after sorting was routinely
?95%. Bone marrow cells were isolated from the femurs and tibias of
tolerant LEW.1A rats.
Quantitative real-time PCR
Quantitative real-time PCR was performed in an Applied Biosystems
GenAmp 7700 sequence detection system using SYBR Green PCR core
reagents. The following oligonucleotides were used: rat iNOS: upper
primer 5?-GACCAAACTGTGTGCCTGGA-3? and lower primer 5?-
TACTCTGAGGGCTGACACAAGG-3?; and rat hypoxanthine phosphori-
bosyltransferase (HPRT): upper primer 5?-CCTTGGTCAAGCAGTA
CAGCC-3? and lower primer 5?-TTCGCTGATGACACAAACATGA-3?.
HPRT was used as an endogenous control gene to normalize varying start-
ing amounts of RNA. Relative expression between a given sample and a
control sample, used for all experiments, was calculated with the
2(???Ct) method. All samples were analyzed in duplicate. Expression of
genes of interest was compared between tolerant animals and syngeneic
Adoptive cell transfer
Cell transfer was performed by i.v. injection into nonirradiated recipients
on the day of kidney transplantation, as previously described (5). Donor
cells were unfractionated spleen cells or MDSC purified from blood and
bone marrow. In other experiments, CFSE-labeled alloreactive T cells were
transferred into irradiated recipients and proliferated in vivo in a graft-vs-
host disease-like manner, as previously described (21). Briefly, 150 ? 106
spleen cells from LEW.1A rats were injected i.v. into allogeneic LEW.1W
recipients that received a sublethal (4.5 Gy) total body irradiation on day
?1. Eight million MDSC from the blood of LEW.1W rats was coinjected.
On day 2.5, spleens were harvested and the number of CFSE?cells was
analyzed by flow cytometry.
Cell culture and proliferation assays
MLRs were performed as previously described (21). Spleen dendritic cells,
used as APC and stimulators, were enriched from LEW.1W spleens by a
14.5% Nicodenz gradient as previously reported (22). APC (105) and re-
sponding cells (105) were cocultured for 5 days in 96-well round-bottom
culture plates in RPMI 1640 medium supplemented with 2 mM L-glu-
tamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% heat-inactivated
FCS, 1% nonessential amino acids, 5 mM HEPES, 1 mM sodium pyruvate,
and 1 ?M 2-ME. Proliferation was measured by addition of 0.5 ?Ci
[3H]thymidine per well. For polyclonal stimulation, T cells were stimulated
with 2.6 ?g/ml anti-CD28 Abs in flat-bottom 96-well plates previously
coated with anti-CD3 (0.5 ?g/ml; 2 h at 37°C). For transwell assays, 3 ?
105cells were placed in the lower and/or upper chamber of a 24-well
Costar Transwell plate (Fisher Scientific) and cultured for 3 days. Prolif-
eration was measured as for MLRs. In suppression assays, NG-monometh-
yl-L-arginine (L-NMMA, Sigma-Aldrich) was used at 5 mM, 1-methyl-D,L-
tryptophan (1-MT; Sigma-Aldrich) was used at 200 ?M, and tin
protoporphyrin (SnPP; Frontier Scientific) at 50 ?M.
Mixed leukocyte reactions
MLRs were performed as previously described (21), except that 5 mM
L-NMMA (Sigma-Aldrich) was added to the medium.
Cytotoxicity was assessed in a51Cr-release assay. Target cells (YAC-1
cells, European Cell Culture Collection) were labeled with51Cr for 60 min
at 37°C in complete medium. Serial dilutions of effector cells in complete
medium were mixed with 300051Cr-labeled target cells in triplicate in
V-bottom 96-well plates and centrifuged for 1 min at 1500 ? g. Plates were
then incubated for 4 h at 37°C, 5% CO2. The supernatants were harvested
(PerkinElmer). Specific cytotoxicity was calculated according to the fol-
lowing formula: (experimental release ? spontaneous release) ? 100/
(maximum release ? spontaneous release).
IFN-? and IL-2 in the supernatants were measured by ELISA using kits
from BD Biosciences and R&D Systems, respectively, according to the
Analyses of cell cytospins were performed after acetone fixation and sat-
uration (using PBS, 4% BSA, 10% goat serum) for 30 min at room tem-
perature. Staining with iNOS Abs (Abcam) was performed overnight at
4°C. Cells were washed 3 times and incubated for 1 h at room temperature
with FITC-conjugated anti-rabbit IgG diluted 1/100 and mounted with
Dako medium (DakoCytomation). Graft samples were embedded in Tissue
Tek OCT compound, snap-frozen in liquid nitrogen, cut into 5-?m sec-
tions, and fixed in acetone. Sections were permeabilized for 30 min at room
temperature using 0.5% saponin, 4% BSA, 2% normal goat serum, 10% rat
serum in PBS, and stained with the primary Ab rabbit anti-iNOS (Abcam),
FITC-conjugated anti-HIS48 mAb, and purified CD11b/c (OX42) mAb
overnight at 4°C. After washing, sections were incubated for 1.5 h with the
secondary Ab Alexa 350-conjugated anti-rabbit (4 ?g/ml) (Invitrogen) and
Alexa 568-conjugated anti-mouse IgG2a (2 ?g/ml; Chemicon Interna-
tional), mounted with Dako medium, and analyzed using a confocal-like
immunofluorescence technique (Zeiss Axiovert 200M microscope with an
7899The Journal of Immunology
by guest on June 4, 2013
Apotome module). Control sections were performed by replacing the pri-
mary Abs with dilution buffer. Naive kidneys were used as negative
Western blot analysis
Cells extracts were prepared in RIPA buffer and quantified by BCA protein
assay reagent (Pierce). Ten micrograms of total protein was resolved by
7.5% SDS-PAGE and transferred onto nitrocellulose membranes (ECL Hy-
bond from GE Healthcare) using a Trans-Blot SD semidry electrophoretic
transfer cell (Bio-Rad). Membranes were blocked with 5% skimmed milk
for 2 h at room temperature and probed overnight at 4°C with 1 ?g/ml
anti-iNOS (Abcam) or 0.1 ?g/ml ?-actin (Santa Cruz Biotechnology) Abs
and with secondary HRP-conjugated goat anti-rabbit Abs (Jackson Immuno-
Research Laboratories) or HRP-conjugated donkey anti-mouse, respec-
tively, diluted 1/2000 in washing buffer. After washing, membranes were
revealed using enhanced chemiluminesence (ECL Western blot, GE
Healthcare) and exposed to Kodak X-Omat LS x-ray films.
Cells were incubated with Alexa 647-conjugated 3.2.3 mAb, PE-conju-
gated CD80 mAb, and biotinylated-conjugated CD86 mAb for 20 min at
4°C. Cells were then washed twice and incubated with streptavidin-PECy7
for 20 min at 4°C. After 15 min saturation, cells were stained for 20 min
with FITC-conjugated mAbs directed against rat CD Ags. To finish, cells
were stained with Alexa 700-conjugated R7-3 or CD6 mAb for 30 min at
room temperature. Analyses were performed using a FACScan II cytoflu-
orometer (BD Biosciences) and FlowJo software. The negative gates were
set using fluorescence-minus-one controls. Apoptosis was assessed by
staining with APC-conjugated annexin V (BD Pharmingen) according to
the manufacturer’s instructions. Evaluation of T cell proliferation by flow
cytometry was performed by staining of pure T cells with 5 ?M CFSE
(Molecular Probes) for 5 min at room temperature and measuring the FL-1
channel after cell culture.
Statistical significance was evaluated using a Mann-Whitney U test for the
comparison of two groups. Graft survival was evaluated by Kaplan-Meier
analysis using the log-rank test.
Regulatory cell phenotype
We have previously demonstrated that administration of anti-
CD28 Abs as an induction treatment results in immune tolerance to
kidney allografts that is characterized by a hyporesponsiveness of
recipient blood cells after challenge with donor alloantigens (4). In
our earlier study, we identified a non-T cell population in the re-
cipient blood that showed suppressive activity and was responsible
for the inhibition of alloreactive T cells. These cells expressed
NKRP-1, CD80, and/or CD86. Herein, we further characterize the
phenotype of these cells. We first noticed that depletion of either
CD80?or CD86?cells from the blood using magnetic beads to-
tally restored the allogeneic proliferative response of PBMC
against donor APC (Fig. 1A), suggesting that the regulatory cells in
this model expressed CD80 and CD86. Next, we analyzed the
phenotype of these cells after gating on non-T (CD6?), CD80/
86?NKRP-1?cells and found them to express CD11a, CD11b,
CD172a (SIRP?), and HIS48 (a rat marker usually associated with
granulocytes (18)). Two subpopulations expressing high and in-
termediate levels of CD11a were observed. However, this varia-
tion in CD11a expression might have been induced by the isolation
procedure (23). These cells additionally expressed class I (OX18)
but not class II (OX6) or CD103 (OX62). Twenty-two percent of
% control PBMC responses
CD6 (T cells)
Class I (RT1A)
Class II (OX6)
from syngeneic kidney graft recipients (open bars) or from tolerant kidney allograft recipients (3 mo posttransplantation; filled bars) were depleted of CD6
(pan-T marker)-negative/CD80?cells, CD6-negative/CD86?cells, or unmodified and stimulated with allogeneic LEW.1W APCs. [3H]thymidine incor-
poration was measured after 5 days. B, Phenotype analysis. PBMC from tolerant kidney allograft recipients (3 mo posttransplantation) were analyzed by
flow cytometry. Cells were gated on CD6?NKRP-1?CD80/86?cells and analyzed for the indicated markers. The percentages of cells expressing each
marker are indicated. Ci, May-Gru ¨nwald Giemsa staining on cytospins of CD6?NKRP-1?CD80/86?cells sorted from PBMC from tolerant kidney allograft
recipients (magnification ?63). Cii, Immunofluorescence analysis of iNOS expressed by CD6?NKRP-1?CD80/86?cells sorted from PBMC from tolerant
kidney allograft recipients (magnification ?40).
Characterization of non-T regulatory cells present in peripheral blood. A, Expression of CD80 and CD86. PBMC of the LEW.1A haplotype
7900Non-T SUPPRESSOR CELLS IN TRANSPLANT TOLERANCE
by guest on June 4, 2013
these cells expressed CD4 and ?10% expressed CD8 (Fig. 1B).
None expressed CD40, CD90, CD25, CD62L, or HIS24 (data not
shown). The absence of class II expression was confirmed by the
fact that depletion of class II-positive cells using magnetic beads
did not restore the alloreactivity of blood cells from the tolerant
recipients (data not shown). After sorting by flow cytometry based
on the CD6?CD80/86?NKRP-1?phenotype, the cells were found
to adhere to plastic culture plates during overnight culture (data not
shown). They presented a homogeneous myeloid-like morphology
with a large, irregularly shaped nucleus and a large cytoplasm
containing inclusions (Fig. 1Ci). These cells were also found to
express iNOS (Fig. 1Cii).
Because depletion of non-T CD80/86?cells from the blood of
tolerant recipients restored the hyporesponsiveness of PBMC
against alloantigens, we measured their suppressive activity after
sorting. In mixed lymphocyte reactions these cells showed a robust
dose-dependent suppressive activity on the proliferation of T cells
stimulated by donor-derived APC (Fig. 2A, dotted bars). More-
over, these cells suppressed the proliferation of anti-CD3 ? anti-
CD28-stimulated T cells (Fig. 2B, open bars). As a control, non-T
CD80/86-negative cells from the blood of the same animals dis-
played no suppressive activity (Fig. 2B, filled bars). The suppres-
sion was maximal 2 and 3 days after initiation of the culture and
partial after 4 days (Fig. 2C). Cells with similar suppressive ac-
tivity could also be isolated from the blood of control recipients of
syngeneic grafts or of naive animals, although in the latter case
fewer cells could be collected (see below for further details). The
suppressive cells isolated from control-transplant recipients or
from tolerant animals had the same suppressive activity on a per
cell basis (Fig. 2A, striped and dotted bars, respectively). Cells
with a phenotype comparable with the one observed in the blood
(i.e., corresponding to the phenotype described in Fig. 1B) could
also be detected in the spleen, lymph nodes, and bone marrow.
These cells isolated from the bone marrow of tolerant recipients
dose-dependently inhibited T cell activation down to a 1:10 ratio,
as did similar cells from the blood. No such suppressive activity,
however, could be measured after sorting from the spleen or lymph
nodes (Fig. 2D).
Accumulation of MDSC in tolerant kidney graft recipients and
function of MDSC in vivo
Given that MDSC from control and tolerant recipients had a
similar suppressive activity on a per cell basis in the suppres-
sion assay (Fig. 2, A and B), we hypothesized that they might
work in vivo as a result of their accumulation. We therefore
compared the number of MDSC in the blood of tolerant allo-
graft recipients to age-matched, syngeneic recipients and found
a significant 2-fold accumulation in tolerant recipients (Fig.
2E). This increase in blood MDSC was not due to the tolerance
induction regimen itself since the administration of anti-CD28
Abs to naive, nontransplant animals failed to result in their
accumulation (data not shown).
To challenge the role of MDSC in vivo, we first tried to transfer
tolerance with MDSC. However, the transfer of 2 ? 106MDSC
isolated from the blood or the bone marrow did not significantly
prolong kidney allograft survival (after the transfer of 2 ? 106
MDSC in three recipients, kidney grafts survived 12, 14, and 16
days vs 11 days in untreated recipients). Transfers repeated on
days 0, 3, and 6 were not more efficient. Additionally, the transfer
of unsorted blood cells (25 ? 106cells) or spleen cells (200 ? 106
cells) had no effect on allograft survival. However, using a previ-
ously described graft-vs-host disease-like system where CFSE-la-
beled T cells are infused into irradiated allogeneic recipients (21),
we observed that coinjection of MDSC prevented the proliferation
of allogeneic T cells in vivo (Fig. 2F).
A, Suppression of mixed lymphocyte reactions. Purified LEW.1A T cells were
stimulated with allogeneic LEW.1W APCs in the presence of the indicated
ratios of effector (“E”) CD6?NKRP-1?CD80/86?cells from the blood of
tolerant allograft recipients or of recipients of syngeneic grafts. Shown are
means ? SD of triplicate wells from one representative experiment out of six.
B, Suppression of polyclonal stimulation. Purified LEW.1A T cells (105cells/
well) were stimulated with anti-CD3/CD28 in the presence of the indicated
ratios of blood CD6?NKRP-1?CD80/86?cells or in the presence of control
blood CD6?NKRP-1?CD80/86?cells from tolerant allograft recipients.
[3H]thymidine incorporation was measured after 3 days. Shown are means ?
SD of triplicate wells from 1 representative experiment out of 10. C, Time-
course of suppression. Same experiment as in B, performed at an E:T ratio of
1:4, where [3H]thymidine incorporation was measured after 2, 3, and 4 days.
D, Immune compartments containing suppressive cells. Purified T cells were
stimulated with anti-CD3/CD28 mAbs in the presence of CD6?NKRP-
nodes. [3H]thymidine incorporation was measured after 3 days. E, Accumu-
lation of MDSC in the blood of tolerant recipients. Blood samples collected on
heparin were cleared of erythrocytes by hypotonic lysis and stained with CD6-
of CD6?NKRP1?CD80/86?MDSC were evaluated in tolerant kidney allo-
graft recipients (filled bar, n ? 5) and in age-matched syngeneic kidney graft
recipients (open bar, n ? 5), 100 days posttransplantation. Results are ex-
pressed as mean cell number/ml blood ? SD. ?, p ? 0.05. Similar results were
found in two other evaluations performed up to day 250 posttransplantation. F,
(150 ? 106) were injected i.v. into irradiated allogeneic rat recipients. Half of
the recipients additionally received 8 ? 106MDSC purified from blood. After
2.5 days, the relative abundance of CFSEhighand CFSElowT cells was mea-
sured in the spleen by flow cytometry. Dotted histogram, controls; open his-
togram, coinjection of MDSC (one figure representative of two independent
Suppressive activity of CD6?NKRP-1?CD80/86?MDSC.
7901 The Journal of Immunology
by guest on June 4, 2013
Mechanisms of action
In the rat, high levels of NKRP-1 expression are characteristic of
NK cells. Because NKRP-1 was expressed on non-T CD80/86?
cells, we tested whether direct cytotoxicity could be responsible
for their suppressive action. Using target YAC-1 cells, we ob-
served an absence of cytotoxic activity, whereas control cells with
the non-T CD80/86-negative NKRP-1?phenotype induced up to
60% cytotoxicity (data not shown), presumably because this cell
population contained NKRP-1highNK cells. Therefore, direct NK-
like cytotoxicity is not involved in the suppressive mechanism of
the non-T CD80/86?NKRP-1?cells. Because NO is known to be
involved in several mechanisms of immunosuppression and be-
cause we detected iNOS expression by immunohistology (Fig.
1Cii), we next asked whether this enzyme also plays a role here. In
suppression assays, a selective inhibitor of iNOS, L-NMMA (used
at 5 mM), was able to reverse the suppression mediated by non-T
CD80/86?NKRP-1?cells (Fig. 3A). The finding that CD11b/c?
cells suppress T cells via NO revealed that these cells had a phe-
notype and mechanism of action compatible with the definition of
MDSC, as defined by Gabrilovitch et al. (12). Therefore, from this
point on, the non-T CD80/86?NKRP-1?cells are referred to as
MDSC. We also noted that 1-methyl-DL-tryptophan (1-MT, used at
200 ?M), an inhibitor of IDO, as well as tin protoporphyrin (SnPP,
used at 50 ?M), an inhibitor of HO-1, were both unable to block
the suppressive activity of MDSC against stimulated T lympho-
cytes (Fig. 3A). Because iNOS was implicated in the suppression
by MDSC, we further tested the level of iNOS expression by West-
ern blot. iNOS was not expressed by freshly isolated blood MDSC,
and resting and activated T cells expressed no or very little iNOS.
Moreover, iNOS was not expressed when MDSC were mixed with
resting T cells. In contrast, after contact between activated T cells
and MDSC, iNOS was strongly up-regulated (Fig. 3B, left section).
Similar experiments were performed using MDSC from the spleen
and lymph nodes (cells that share a comparable phenotype were
isolated from these compartments). In this case, however, no iNOS
up-regulation was observed in any of the conditions tested (data
To determine whether cell contact between MDSC and target T
cells was required for suppression, we performed transwell assays.
Stimulated LEW.1A T cells were placed in the lower chamber with
MDSC isolated from LEW.1A-tolerant kidney graft recipients in
the upper chamber of the transwell. The physical separation abro-
gated the suppression, revealing a contact-dependent inhibition of
the proliferative response (Fig. 4). Moreover, the physical separa-
tion from MDSC reduced the suppression even when the MDSC
were mixed with other activated T cells, suggesting that activated
T cells must be in contact with MDSC not only to elicit suppres-
sion but also to become sensitive to suppression (Fig. 4, lower
bar). With the aim of identifying molecular interactions between
MDSC and activated T cells that might be required for suppres-
sion, we tested several antagonistic Abs in the suppression assays.
However, no modification of suppression could be obtained with
Abs against CD80, CD86, CD80 ? CD86 (tested at 10 ?g/ml and
at 50 ?g/ml), class I, class II, CD11b/c, IFN-?, CD172a, CD40,
IL-4, or IL-10 (tested at 10 ?g/ml; data not shown). To further
understand how MDSC and NO blocked T cell proliferation, we
analyzed their possible proapoptotic effect. Proliferation and apo-
ptosis were measured using double staining with CFSE and an-
nexin V after 2 days of culture. As shown in Fig. 5Ai, CFSE-
labeled T cells proliferated after polyclonal stimulation and 40% of
them were apoptotic. In the presence of MDSC, proliferation was
minimal and 76.2% of the cells were found to be apoptotic. Thus,
MDSC seem to affect the viability of stimulated T cells by block-
ing their proliferation and by inducing apoptosis in a contact-de-
pendent manner. In similar assays, we also noticed that despite
inhibiting T cell proliferation, MDSC only moderately prevented T
cell activation after polyclonal stimulation, because ?60% of them
MDSC. A, Role of iNOS in the suppression. T cells from naive animals
were stimulated with anti-CD3/CD28 Abs in the presence of a 1:3 E:T ratio
of control CD6?NKRP-1?CD80/86?cells or CD6?NKRP-1?CD80/86?
MDSC extracted from the blood of tolerant kidney graft recipients, added
to inhibit proliferation. Enzyme inhibitors were added to the cultures (L-
NMMA, iNOS inhibitor; SnPP, HO-1 inhibitor; 1-MT, IDO inhibitor). Pro-
liferation was measured after 3 days by [3H]thymidine incorporation. Data
are mean cpm ? SD of one representative experiment out of three. B,
Western blot analysis of iNOS expression. Cells were cultured as in A for
2 days. Whole protein (10 ?g) from the indicated cultures were resolved on
a 7.5% SDS-PAGE containing 10 mM DTT and blotted onto nitrocellulose
filters. Membranes were hybridized with anti-iNOS and actin Abs plus
secondary Ab and revealed by chemiluminescence.
Mechanisms of actionof CD6?NKRP-1?CD80/86?
bers were used to prevent direct cell contact between anti-CD3/CD28-
stimulated T cells (6 ? 105cells from naive animals) and MDSC or control
CD6?CD80/86?cells (1.5 ? 105cells from the blood of tolerant kidney
graft recipients). Proliferation was assessed in the lower chamber by
[3H]thymidine incorporation after 3 days of culture. Data are mean cpm ?
SD of triplicate wells in two independent experiments.
MDSC act in a contact-dependent manner. Transwell cham-
7902 Non-T SUPPRESSOR CELLS IN TRANSPLANT TOLERANCE
by guest on June 4, 2013
expressed CD25 and most had lost their expression of CD62L (Fig.
5, Aii and Aiii).
Differential effect on effector T cells and Tregs
According to the CFSE dilution assays after polyclonal activation,
although MDSC blocked the proliferation of most CD25?T cells,
a subpopulation of CD25highT cells escaped suppression (Fig.
5Aii, arrow), suggesting that CD25?Tregs might not be sensitive
to MDSC. To directly measure this effect, we sorted CD4?CD25?
effector and CD4?CD25high
regulatory cells (93% of the
CD4?CD25highcells expressed FoxP3 in this assay) and tested the
action of MDSC on the proliferation of these cell subpopulations.
Effector T cells proliferated strongly after 3 days, and their pro-
liferation could be fully inhibited by MDSC. In contrast, MDSC
inhibited the proliferation of CD4?CD25highFoxP3?Tregs by
50% only (Fig. 5B). The cytokines secreted by these two T cell
populations were also differentially affected by MDSC: stimulated
CD4?CD25?effector T cells produced less IFN-? in the presence
of MDSC, an observation compatible with the inhibition of their
proliferation. In contrast, IL-2 production by stimulated effector T
cells was not abolished by MDSC but rather was enhanced (Fig.
5Ci). Again, this is compatible with the idea that T cell prolif-
eration, but not activation, is blocked by MDSC (IL-2 would
therefore be released and, since not consumed by T cells, ac-
cumulate in the medium). As previously described (24), stim-
ulated CD4?CD25highFoxP3?Tregs produced very low quan-
tities of IL-2 and produced some IFN-? that was enhanced by
the addition of MDSC (Fig. 5Cii).
C i Effector T cells
ii Regulatory T cells
0 10000 20000 30000
Effector T cells
Anti CD3/CD28 stimulated
T CD4+CD25- cells
Anti CD3/CD28 stimulated
T CD4+CD25highFOXP3+ cells
anti-CD3/CD28-stimulated CFSE-labeled T cells with blood MDSC or control cells (CD6?CD80/86?) for 2 days, DAPI-negative cells were stained
with annexin V (i), APC-conjugated CD25 (ii, arrow pointed on CD25highcells), or APC-conjugated CD62L (iii) and analyzed by flow cytometry.
In these analyses, a threshold for CFSE fluorescence was set at a value of 100 to exclude CFSE-negative cells from the evaluation. B, Differential
effect of MDSC on regulatory vs effector T cell proliferation. Spleen T cells were sorted into Tregs and effector T cells according to their
CD4?CD25high(these cells were mostly FoxP3?) or CD4?CD25?(FoxP3?) phenotypes. Each population was stimulated with anti-CD3/CD28 and
cultured with or without MDSC (ratio of 5 target cells for 1 MDSC) for 3 days. Proliferation was measured after 3 days by incorporation of
[3H]thymidine. Results are means ? SD of triplicate wells and representative of two experiments. C, Differential effect of MDSC on cytokine
synthesis by regulatory vs effector T cells. Same experiment as in B, where supernatants were collected after 48 h. i, CD4?CD25?effector T cells.
ii, CD4?CD25highregulatory T cells. IFN-? and IL-2 production was measured by ELISA. Results are means of triplicate measurements ? SD from
one experiment representative of four.
Effector function of MDSC. A, MDSC block proliferation but only moderately block activation of T lymphocytes. After coculture of
7903 The Journal of Immunology
by guest on June 4, 2013
Graft infiltration and expression of iNOS
By immunohistology, we found CD11b?cells in the glomeruli of
control and tolerated grafts (Fig. 6, Aa and Ab). In the parenchyma,
CD11b?cells (Fig. 6Ab) synthesizing iNOS were found only in
tolerant allografts (Fig.6Ae–f).
CD11b?HIS48?iNOS?cells could be detected in the parenchyma
and associated with the blood vessel walls (Fig. 6, Ab and Ac–f).
Moreover, a quantitative analysis of messenger RNA for iNOS
revealed a significantly higher expression in tolerated grafts than in
syngeneic grafts (Fig. 6B). Also, more iNOS mRNA was visible in
the blood of tolerant recipients (Fig. 6B).
At higher magnification,
Role of MDSC in transplant tolerance
Because the tolerated kidney grafts were infiltrated with CD11b
cells expressing iNOS (Fig. 6A) and accumulated more iNOS
mRNA (Fig. 6B), it is likely that in tolerant recipients, MDSC
accumulate and localize in the graft. To challenge the hypothesis
that tolerance was achieved as a result of the activity of the iNOS
enzyme, we tested the effect of injection of aminoguanidine, an
inhibitor of iNOS, on tolerance 120 days after kidney transplan-
tation. The results showed that tolerant recipients rejected their
graft within an average of 10 days after aminoguanidine injection
(100 mg/kg i.p. twice daily; Fig. 7). A pathological examination of
these grafts revealed that acute, cellular-mediated rejection was the
origin of the graft failure. These data demonstrate that the main-
tenance phase of tolerance in this model requires an active syn-
thesis of NO.
Herein, we show that the rat model of anti-CD28 Ab-induced
kidney allograft tolerance triggers the accumulation of plastic-
adherent CD11b?myeloid cells expressing CD80/86 that can
be defined as MDSC. In vitro, these cells induced a contact-
dependent apoptosis of activated effector T cells that them-
selves triggered the expression of iNOS by MDSC. MDSC had
a limited effect on the proliferation of CD4?CD25highFoxP3?
Tregs that failed to induce iNOS in MDSC. The action of NO
production was critical to the immunosuppression mediated by
MDSC and in maintaining the tolerant state in vivo.
It has become clear that transplant tolerance uses multiple cel-
lular mechanisms that cooperate to suppress immunity, involving
several types of regulatory T cells and tolerogenic DCs. Cooper-
ation between different cell types might even be required to estab-
lish infectious tolerance to kidney allografts (5). In these situa-
tions, it is thought that CD152 up-regulated on Tregs interacts with
CD80 on tolerogenic DCs in an Ag-cognate manner. On the one
hand, this interaction results in the maintenance of Treg suppres-
sive activity and, on the other hand, promotes a CD80-dependent
(and IFN-?-dependent) up-regulation of IDO, an enzyme that de-
grades the essential amino acid tryptophan (6). Tryptophan me-
tabolites then suppress T cell responses as well as T cell clonal
expansion. In vivo, CTLA-4/CD80 (and/or CD86) interactions
have been shown to be required for tolerance after heart allo-
transplantation in mice (25), as well as IDO activity after heart
allotransplantation in the rat (3). Tumors can also modulate
immune responses by triggering an immune tolerance. In the latter
case, although IDO induced by the action of local Tregs contrib-
utes to tumor-induced tolerance (26), the accumulation of MDSC
appears to be a dominant mechanism in rodents as well as in man
(10). The mechanism of action of MDSC typically involves the
synthesis of NO (13) and/or the action of arginase 1 (27). Addi-
tionally, previous studies have suggested that mice Gr-1?CD11b?
MDSC express CD80 and suppress immune responses to tumors
by promoting IDO up-regulation after engagement of CTLA-4 ex-
pressed by infiltrating activated or regulatory T cells (16). Herein,
we show that the MDSC that accumulate in kidney allograft tol-
erance also express CD80. However, in our hands, CD80-CD152
interactions were not essential factors for MDSC function in vitro,
because anti-CD80 Abs had no effect on the system. Nevertheless,
the removal of CD80?cells as well as CD86?cells from the
MLRs restored proliferation, suggesting that MDSC do express
both markers. In addition, MDSC-mediated suppression in vitro
was IDO independent, because it was not reversed by the IDO
inhibitor 1-MT. As a comparison, 1-MT was shown to reverse the
suppression driven by tolerogenic DC and by CD11b?monocytes
tological analysis of kidney grafts. Syngeneic grafts (a) and tolerated al-
lografts (b) stained with CD11b (red fluorescence) and HIS48 (green flu-
orescence) Abs. Magnification ?10. The arrows indicate blood vessel
sections. c–f, tolerated kidney allograft, at a magnification of ?40, focused
on a blood vessel section, stained for HIS48 (c, green fluorescence), iNOS
(d, blue fluorescence), CD11b (e, red fluorescence), and merged staining
(f). B, Assessment of iNOS mRNA. The level of iNOS mRNA was ana-
lyzed by quantitative PCR in kidney grafts and blood from syngeneic or
tolerant recipients 100 days after transplantation. ?, p ? 0.05; ??, p ? 0.01.
Graft infiltration and expression of iNOS. A, Immunohis-
ney allograft recipients (?120 days posttransplantation) received i.p. in-
jections of 30 mg/kg of the iNOS inhibitor aminoguanidine (AG) every
12 h. Recipient survival is represented. Control rats also received amino-
guanidine, and kidney function was recorded twice a week for 20 days. In
these controls, kidney function was unmodified (uremia of 4 mmol/L and
creatininemia of 18 ?mol/L).
iNOS inhibition in vivo brakes tolerance. Four tolerant kid-
7904 Non-T SUPPRESSOR CELLS IN TRANSPLANT TOLERANCE
by guest on June 4, 2013
(4), which are different from the MDSC described here, in the
same rat strain combination (5). Instead, in vitro, we found that the
immunosuppressive activity of rat MDSC was solely controlled by
NO. The fact that iNOS was detectable in isolated MDSC as well
as in graft-infiltrating cells reinforces this idea. In vivo, the fact
that injection of the iNOS inhibitor aminoguanidine induced the
rejection of otherwise tolerated allografts also showed that the
maintenance of the tolerant state was under the control of NO and
not of IDO. In other tolerance models with the same rat strain
combination, however, inducing rejection of tolerated allografts
necessitated the administration of both IDO and iNOS inhibitors
(28), indicating the possible coexistence of two mechanisms that
cooperate to maintain transplant tolerance. Therefore, our data
point toward a functional difference between mouse Gr-
1?CD11b?MDSC that mediate suppression via NO and possibly
IDO and the rat MDSC described herein that appear not to use the
In mice, MDSC function is also dependent on IFN-? (29). In our
rat system in vitro, we did not find a critical role for this factor,
because anti-IFN-? Abs failed to modify the suppression of MDSC
on anti-CD3 ? anti-CD28-activated T lymphocytes. Because
SIRP? is an inhibitory receptor that modulates macrophage and
DC function (30) and because it was expressed by rat MDSC, it
was possible that SIRP?-CD47 interactions reinforced the sup-
pressive activity of MDSC. However, the suppression by MDSC
was not reduced by anti-SIRP? Abs either. Preventing CD40-
CD40L molecular interactions or the action of IL-4 and IL-10
cytokines was also inefficient. Thus, the interactions required for
MDSC function in the rat require further exploration.
Our investigations suggest that the MDSC-mediated suppres-
sion lacks Ag specificity, because MDSC could regulate the pro-
liferation of third-party APC-stimulated T cells as well as the
proliferation of anti-CD3 ? anti-CD28-stimulated T cells. Addi-
tionally, MHC class II expression was not detected by flow cy-
tometry and depleting MHC class II?cells from the blood did not
prevent T cell unresponsiveness in the MLRs. Thus, it appears that
MDSC do not interact with CD4?T cells in a cognate manner.
Therefore, the capacity of MDSC to generate suppressive signals
when encountering activated T cells most likely serves to regulate
immune responses during times of heightened immune activity,
without Ag specificity. In transplantation, MDSC might have a
regulatory function, in cooperation with other, Ag-specific, regu-
latory cells. Herein, the observation that inhibition of iNOS in vivo
induced rejection suggests that NO-based suppression mechanisms
are not dispensable.
An important issue was the location in vivo where a contact
could occur between effector T cells and blood MDSC. We de-
tected MDSC in the blood of tolerant recipients of kidney allo-
grafts. In the spleen and lymph nodes, cells were identified that had
a comparable phenotype but were devoid of ex vivo suppressive
activity. By immunohistology, infiltrating cells expressing CD11b,
HIS48, and iNOS markers could be detected in tolerated allografts,
in the parenchyma, as well as being associated with blood vessel
walls (CD80 and CD86 could not be detected by immunohistology
in the rat species because the Abs do not bind to fixed tissues).
Although we were unable to prove that these cells have a suppres-
sive activity in situ, this phenotype is compatible with the presence
of MDSC within the graft. Additionally, messenger RNA for iNOS
was found to be accumulated 4-fold in tolerated kidneys. It is
therefore possible that blood MDSC suppress T cells inside the
graft. This mechanism of action would then be similar to tumor-
infiltrating MDSC. In this kidney transplant model, however, tol-
erance is clearly associated with the control, and not the elimina-
tion, of alloreactive T cells, because they can be detected in the
periphery several months after transplantation. Indeed, the simple
removal of MDSC in vitro from blood cells collected on day 100
was sufficient to lift the suppression of lymphocyte alloreactivity,
indicating that the Ag-specific lymphocytes had not been deleted
in tolerant recipients. We have previously reported that donor-
specific alloreactive T lymphocytes in the blood of tolerant recip-
ients of kidney allografts expressed high rates of activation and
apoptotic markers (4). This suggested that alloreactive T cells were
continuously produced, presumably as a result of a thymic output,
and kept under control by contact with MDSC, which as a result
induced apoptosis not only in the graft but also in the blood.
Herein, we confirmed in vitro that reactive T cells express activa-
tion markers (express CD25 and lose CD62L) and undergo apo-
ptosis at a high rate in the presence of MDSC.
The question remains as to what extent MDSC participate to
tolerance induction or maintenance in vivo. Although the direct
evaluation of their role would be provided by an adoptive transfer
of MDSC, correlated with tolerance, none of our trials including
transfer of spleen cells, blood cells, blood MDSC, and bone mar-
row MDSC could induce tolerance. Instead, we found a slight,
nonsignificant delay in the occurrence of rejection after transfer of
blood or blood-derived MDSC. One reason might be that MDSC
lose their suppressive activity after transfer, possibly as a result of
differentiation. Another might be that NO could play a role in the
maintenance phase but not in the induction phase of the tolerance
that might be under the control of other mechanisms such as IDO,
as previously shown in transplantation trials in the same rat strain
combination (3, 28).
A challenging observation was that MDSC were also present in
recipients of isografts as well as in naive animals. MDSC from
control animals appeared to have a similar activity to those from
tolerant recipients on a per cell basis. Therefore, the only differ-
ence was the increase in numbers of these cells in tolerant recip-
ients. These features characterize MDSC as natural modulators of
immune reactivity, mobilized by tumors, but also by tolerated al-
lografts, to establish or reinforce tolerance. This view is strength-
ened by the recent observation that genetic inactivation of CD11b
abolishes oral tolerance without compromising APC maturation or
Ag-specific immune activation (31), establishing a specific role of
CD11b?cells in oral tolerance induction.
A novel finding was that MDSC do express iNOS upon contact
with activated T cells, but not upon contact with activated Tregs.
Moreover, MDSC block the expansion of effector T cells and, to a
lesser extent, CD4?CD25?FoxP3?Tregs. The fact that MDSC
spare Tregs directly reinforces the suggestion by Yang et al. (16)
that MDSC might mediate suppression at least in part via Tregs.
However, in our study MDSC suppressed the proliferation of stim-
ulated CD4?CD25?T lymphocytes to the same extent as unsorted
T cells, clearly showing that the presence of CD4?CD25?FoxP3?
Tregs is not required, at least in vitro, for MDSC-mediated
In summary, our studies show a significant accumulation of
MDSC in a rat model of kidney transplant tolerance. These cells
have a nonspecific immunosuppressive activity in vivo and in
vitro, involving the action of iNOS, which is up-regulated after
contact with activated effector T cells but not with Tregs. These
data illustrate a novel immunoregulatory mechanism associated
with transplant tolerance.
We are grateful to S. Brouard, R. Josien, and J. Ashton-Chess for critically
reading the manuscript, and we thank P. Hulin and the Institut National de
la Sante ´ et de la Recherche Me ´dicale-IFR26 confocal microscopy platform.
7905The Journal of Immunology
by guest on June 4, 2013
Disclosures Download full-text
The authors have no financial conflicts of interest.
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