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Function of CD4+CD25+ Treg cells in MRLlpr mice is compromised by intrinsic defects in antigen-presenting cells and effector T cells.

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ARTHRITIS & RHEUMATISM
Vol. 58, No. 6, June 2008, pp 1751–1761
DOI 10.1002/art.23464
© 2008, American College of Rheumatology
Function of CD4⫹,CD25⫹ Treg Cells in
MRL/lpr Mice Is Compromised by Intrinsic Defects in
Antigen-Presenting Cells and Effector T Cells
Véronique Parietti, Fanny Monneaux, Marion Décossas, and Sylviane Muller
Objective. Naturally occurring CD4ⴙ,CD25ⴙ
Treg cells are central in the maintenance of peripheral
tolerance. Impaired activity and/or a lower frequency of
these cells is involved in the emergence of autoimmunity. We undertook this study to analyze relative proportions and functional alterations of Treg cells in
MRL/lpr mice.
Methods. The frequency of CD4ⴙ,CD25ⴙ T cells
in the peripheral blood of healthy and autoimmune
mice was compared by flow cytometry. The capacity of
CD4ⴙ,CD25ⴙ T cells to inhibit the proliferation and
cytokine secretion of CD4ⴙ,CD25– T cells was assessed
after polyclonal activation.
Results. MRL/lpr mice exhibited a normal percentage of CD4ⴙ,CD25high T cells, and forkhead box P3
messenger RNA and protein expression in Treg cells
was not altered. However, MRL/lpr Treg cells displayed
a reduced capacity to suppress proliferation and to
inhibit interferon-␥ secretion by syngeneic effector
CD4ⴙ,CD25ⴚ T cells, as compared with syngeneic
cocultures of CBA/J T cells. Moreover, effector MRL/lpr
CD4ⴙ,CD25ⴚ T cells were substantially less susceptible to suppression even when cultured with CBA/J or
MRL/lpr Treg cells. Crossover experiments led us to
conclude that in MRL/lpr mice, each partner engaged in
T cell regulation displays altered functions. Molecules
involved in suppressive mechanisms (CTLA-4 and
CD80/CD86) are underexpressed, and antigenpresenting cells (APCs) produce raised levels of
interleukin-6, which is known to abrogate suppression.
Conclusion. Our results suggest that although the
frequency and phenotype of Treg cells in MRL/lpr mice
are similar to those in normal mice, Treg cells in
MRL/lpr mice are not properly stimulated by APCs and
are unable to suppress proinflammatory cytokine secretion from effector T cells.
Naturally arising CD4⫹ Treg cells expressing the
interleukin-2 receptor ␣-chain (CD25) (1) and the transcription factor forkhead box P3 (FoxP3) represent a
subset of thymus-derived CD4⫹ T cells that is critical for
the control of most immune responses, including autoimmunity, transplantation tolerance, antitumor immunity, and antiinfectious responses. Following engagement of their T cell receptor (TCR), Treg cells
expressing the highest levels of CD25 (CD4⫹,CD25high)
are thought to suppress the proliferation and activity of
conventional CD4⫹,CD25– effector T cells as well as
CD8⫹ T cells. The mechanisms by which Treg cells
mediate their suppressive effect in vivo have not been
fully elucidated. In vitro, Treg cells are able to inhibit the
proliferative response of CD4⫹ and CD8⫹ T cells by
different mechanisms that are dependent on cell–cell
contact and independent of the production of soluble
molecules such as transforming growth factor ␤ and
IL-10 (2). The role in suppression of Treg cell–expressed
molecules such as CTLA-4, glucocorticoid-induced tumor necrosis factor (TNF) receptor, lymphocyte
function–associated antigen 1/CD18, ␣E␤7 integrin/
CD103, and L-selectin/CD62L (3–5) was investigated,
but the evidence for their involvement remains circumstantial (6). Non–T cells can also be censored by
CD4⫹,CD25high Treg cells, and it has been shown that
the latter modulate the maturation of human blood
myeloid, but not plasmacytoid, dendritic cells (7) and
Supported by the CNRS. Ms Parietti’s work was supported by
a grant from the French Association de Recherche sur la Polyarthrite.
Dr. Monneaux was recipient of a prize from the French Fondation
pour la Recherche Médicale, Paris.
Véronique Parietti, BSc, Fanny Monneaux, PhD, Marion
Décossas, PhD, Sylviane Muller, PhD: CNRS, Institut de Biologie
Moléculaire et Cellulaire, Strasbourg, France.
Address correspondence and reprint requests to Sylviane
Muller, PhD, Institut de Biologie Moléculaire et Cellulaire, UPR 9021,
CNRS, 15 rue René Descartes, 67000 Strasbourg, France. E-mail:
S.Muller@ibmc.u-strasbg.fr.
Submitted for publication October 1, 2007; accepted in revised form February 15, 2008.
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can act directly on B cells by inducing B cell death in a
cell contact–dependent manner (8).
FoxP3⫹,CD4⫹,CD25⫹ Treg cells are regarded
with great interest because they are also present in T
cell–B cell area borders and within germinal centers of
human lymphoid tissues and can directly suppress B cell
Ig production and class switch recombination without
having to suppress T cells (9). Treg cell depletion can
lead to aberrant antibody production (10), and administration of Treg cells into autoimmune animals can
significantly reduce autoantibody response (11). Thus, in
several autoimmune diseases in which autoantibodies
are produced, the function or the number of Treg cells is
decreased (10,12–14).
Although there are still some discrepant reports
(possibly due to variations in CD4⫹,CD25⫹ T cell
analysis), studies in patients with diabetes, multiple
sclerosis, rheumatoid arthritis (RA), type II autoimmune polyendocrinopathy, psoriasis, or systemic
lupus erythematosus (SLE) have shown that Treg cell
suppressive functions tested ex vivo are compromised
(15–22). Other studies in lupus, primary Sjögren’s syndrome, and RA demonstrated no functional impairment of CD4⫹,CD25high Treg cells (23–25). However,
reduced numbers of CD4⫹,CD25⫹ T cells in the peripheral blood of SLE patients have been described
(23,26–28).
Lupus-prone mouse models offer the possibility
of examining more precisely, and at different stages of
the disease, the characteristics of FoxP3⫹,
CD4⫹,CD25high Treg cells without interference from
immunosuppressive or glucocorticoid treatments (29).
Previous studies have revealed a numerical deficiency
of CD4⫹,CD25⫹ Treg cells in (NZB ⫻ NZW)F1 and
(SWR/NZB)F1 lupus-prone mice (30). The functionality
of (NZB ⫻ NZW)F1 Treg cells was also explored, and
no intrinsic defect in suppressive function of thymusderived Treg cells was found in these mice (31). In vitro,
only marginal alteration of Treg cell function was observed in MRL/Mp mice (32), in which a modest lupus
disease develops.
In the present study, we examined the frequency
and functional properties of CD4⫹,CD25⫹ Treg cells
in MRL/lpr mice, in which a strong lupus disease develops. We found that compared with nonautoimmune
mice, MRL/lpr mice exhibit normal percentages of peripheral CD4⫹,CD25high T cells, and that FoxP3 messenger RNA (mRNA) and protein expression in
CD4⫹,CD25⫹ T cells is not altered. However, peripheral MRL/lpr CD4⫹,CD25high T cells display a re-
PARIETTI ET AL
duced capacity to suppress proliferation and, especially,
to inhibit interferon-␥ (IFN␥) secretion by syngeneic
effector CD4⫹,CD25– T cells. We further observed that
effector MRL/lpr CD4⫹,CD25⫺ T cells are significantly
less susceptible to suppression. Mechanistically, we
found that in MRL/lpr mice, CD80/CD86 and CTLA-4
(which are required for suppression) are underexpressed
on effector T cells and antigen-presenting cells (APCs)
and on Treg cells, respectively, and that IL-6, which is
known to abrogate suppression, is overproduced by MRL/
lpr APCs.
MATERIALS AND METHODS
Mice. Female BALB/c (H-2d), CBA/J (H-2k), MRL/Mp
(H-2k), and MRL/lpr (H-2k) mice were purchased from Harlan
(Gannat, France). Animal experiments were reviewed and
approved by the Regional Ethics Committee of Strasbourg
(CREMEAS, project no. AL/09/12/03/07).
Antibodies. Fluorescein isothiocyanate–labeled antiCD25 (7D4), phycoerythrin (PE)–labeled anti–CD62 ligand
(anti-CD62L) (MEL-14), PE-labeled anti-CD4 (RM4.5), PElabeled anti-CD80 (16-10A1), PE-labeled anti-CD86 (GL1),
PE-labeled anti–CTLA-4 (UC10-4F10-11), PE-labeled Armenian hamster IgG2 (isotype control), PE-labeled rat IgG2a
(isotype control), peridinin chlorophyll protein–labeled anti-CD4,
allophycocyanin-labeled anti-CD4, allophycocyanin-labeled
anti-B220 (RA3-6B2), and purified anti-CD3 (145-2C11)
monoclonal antibodies (mAb) were purchased from BD Biosciences (San Diego, CA). Allophycocyanin-labeled anti-FoxP3
mAb were purchased from eBioscience (San Diego, CA).
Flow cytometry. To determine the proportion of
CD4⫹,CD25⫹ T cells in the peripheral blood, 50 ␮l of whole
blood was incubated at 4°C in phosphate buffered saline
containing 2% fetal calf serum (FCS) (PAN; Dutscher, Brumath, France) with the fluorescent antibodies followed by
FoxP3 intracellular staining using a FoxP3 staining kit (eBioscience). Cells were acquired and analyzed with a FACSCalibur flow cytometer using CellQuest research software (BD
Biosciences). Flow cytometry analysis of FoxP3 intracellular
expression was also performed using purified CD4⫹,CD25⫹
T cells and a FoxP3 staining kit. Staining for intracellular
CTLA-4 was performed using the Fixation/Permeabilization
Solution Kit (BD Biosciences).
Cell preparation. CD4⫹,CD25⫺ and CD4⫹,CD25⫹
T cells were purified from lymph node (LN) cells. Briefly, LN
cells from healthy CBA/J, MRL/Mp, and autoimmune-prone
MRL/lpr mice were enriched for CD4⫹ T cells by negative
selection. LN cells were depleted of macrophages, granulocytes, B cells, and CD8⫹ T cells by incubation with anti-CD11b
(Mac-1), anti-GR1 (8C5), anti-CD19 (1D3), anti-B220 (6B2),
and anti-CD8 (Lyt-2) mAb purified in-house and with magnetic beads coupled to anti-rat Ig (Dynal, Oslo, Norway).
CD25⫹ cells were isolated from the CD4⫹ cell population by
staining with PE-labeled anti-CD25 mAb followed by incubation with magnetic-activated cell sorting (MACS) anti-PE
microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
IMPAIRMENT OF TREG CELLS IN MRL/lpr MICE
1753
Figure 1. Frequency of peripheral CD4⫹,CD25⫹ Treg cells and forkhead box P3 (FoxP3) expression in MRL/lpr and normal mice. A, Analysis of
CD4⫹,CD25high,CD62 ligand (CD62L)⫹,FoxP3⫹ T cells (Treg cells) was performed in the peripheral blood of 4 healthy BALB/c mice, 4 healthy
CBA/J mice, and 6 lupus MRL/lpr mice. Whole blood was stained for CD4, CD25, CD62L, and FoxP3. The percentage of Treg cells in the total
CD4⫹ T cell population was determined by fluorescence-activated cell sorting analysis. Bars show the mean and SD. B, FoxP3 expression in the
subpopulations of CD4⫹ T cells is shown. Magnetic-activated cell–sorted CD4⫹,CD25⫺ and CD4⫹,CD25⫹ T cells were purified from lymph nodes
of BALB/c, CBA/J, and MRL/lpr mice. After mRNA extraction and reverse transcription, cDNA from each population was subjected to
semiquantitative polymerase chain reaction using FoxP3- and hypoxanthine guanine phosphoribosyltransferase (HPRT)–specific primers. C,
Intracellular expression of FoxP3 protein in BALB/c, CBA/J, and MRL/lpr mice was assessed by flow cytometry on purified CD4⫹,CD25⫹ T cells
used for functional studies. The percentages of FoxP3⫹ cells among total CD4⫹,CD25⫹ T cells are indicated.
CD4⫹,CD25⫹ T cells were then positively selected on a
MACS mini-separation magnetic column, and the flowthrough fraction containing CD4⫹,CD25⫺ T cells was collected. The purity of cell subsets was ⬎95% as determined by
fluorescence-activated cell sorting analysis. For analysis of
CTLA-4 expression, CD25⫹ cells were purified from the
CD4⫹ cell population by staining with biotin-conjugated antiCD25 mAb followed by incubation with MACS antibiotin
microbeads (Miltenyi Biotec).
For in vitro proliferative assays, the stimulation was
performed by T cell–depleted splenic APCs. Briefly, after red
blood cell lysis, splenocytes were incubated with anti-CD4
(GK1.5) and anti-CD8 (Lyt-2) mAb and with magnetic beads
coupled to anti-rat Ig. T cell–depleted APCs (consisting mainly
of B cells) were then treated with mitomycin C (500 ␮g/ml;
Sigma-Aldrich, St. Louis, MO).
Polyclonal activation and suppression assay. CD4⫹,
CD25⫺ T cells were cultured (105/well) in plates precoated
with anti-CD3 mAb (1 ␮g/ml for proliferative assays, 2 ␮g/ml
for cytokine secretion) in the presence of APCs (5 ⫻ 104/well
T cell–depleted and mitomycin C–treated splenic cells), with
or without CD4⫹,CD25⫹ T cells (105/well). Cultures were
performed in RPMI 1640 medium (Cambrex, Verviers, Belgium) supplemented with 10% FCS, 10 ␮g/ml gentamicin
(Cambrex), 10 mM HEPES (Cambrex), and 0.05 mM
2-mercaptoethanol. Culture supernatants were collected after
48 hours and tested for IFN␥, IL-2, and IL-6. To measure cell
proliferation, 3H-thymidine (1 ␮Ci; specific activity 6.7 Ci/
mmole) was added after 48 hours of culture, cells were
harvested 18 hours later on a filter with an automatic cellharvesting device (Packard, Meriden, CT), and thymidine
incorporation was assessed by using a Matrix 9600 direct beta
1754
PARIETTI ET AL
counter (range 10–35,000 counts per minute; Packard). Data
were expressed as the percentage of inhibition, calculated as
follows: % inhibition ⫽ [1 ⫺ (cpm of CD4⫹,CD25⫺ plus
CD4⫹,CD25⫹)/(cpm of CD4⫹,CD25⫺)] ⫻ 100. Proliferation
experiments were performed in triplicate. In some assays,
anti–IL-6 mAb (MP5-20F3; BD Biosciences) was added to
neutralize IL-6 (5 ␮g/ml).
IL-6 secretion assay. To assess levels of IL-6 secretion,
T cell–depleted splenic APCs (5 ⫻ 105/well) were cultured
with increasing concentrations of lipopolysaccharide (LPS;
Sigma-Aldrich). The supernatants were harvested after 24
hours of culture and stored at ⫺20°C until tested for IL-6
levels.
Cytokine analysis. The levels of IL-2, IL-6, and IFN␥
in the culture supernatants were determined using capture
enzyme-linked immunosorbent assays according to the instructions of the manufacturer (BD Biosciences). Results were
expressed as the cytokine concentration in pg/ml. The detection limit was 15 pg/ml for IL-2 and IFN␥ and 30 pg/ml for
IL-6.
Polymerase chain reaction (PCR). Total RNA was
extracted from CD4⫹,CD25⫹ T cells and CD4⫹,CD25⫺ T cells
using TRI Reagent (Sigma-Aldrich). Complementary DNA
(cDNA) was synthesized from 1 ␮g of each RNA sample using
the RevertAid First Strand cDNA Synthesis Kit (Fermentas,
Burlington, Ontario, Canada). PCR was performed as previously described (33). The relative quantity of cDNA in each
sample was normalized by semiquantitative PCR for hypoxanthine guanine phosphoribosyltransferase (HPRT). Primer
sequences for FoxP3 were as follows: 5⬘-CAGCTGCCTACAGTGCCCCTAG-3⬘ (forward) and 5⬘-CATTTGCCAGCAGTGGGTAG-3⬘ (reverse).
Statistical analysis. Analysis for statistically significant
differences was performed with Student’s t-test. P values less
than 0.05 were considered significant.
RESULTS
Normal frequency of CD4ⴙ,CD25ⴙ Treg cells
displayed by MRL/lpr lupus mice. In order to determine whether the frequency of Treg cells is affected in
MRL/lpr mice, we measured the percentages of
CD4⫹,CD25high,CD62L⫹,FoxP3⫹ T cells in the blood
of 6 female MRL/lpr mice, 4 female BALB/c mice, and
4 female CBA/J mice at different ages. As shown in
Figure 1A, the proportion of this cell subset among
peripheral CD4⫹ T cells decreased with aging (2.4% at
8 weeks and 1.7% at 17 weeks, on average), and this was
similar in MRL/lpr, CBA/J, and BALB/c mice. We next
studied expression of the transcription factor FoxP3 at
the level of both mRNA and protein. As expected,
LN CD4⫹,CD25⫺ T cells from the 3 mouse strains did
not express FoxP3 mRNA, while CD4⫹,CD25⫹ T cells
did (Figure 1B). The levels of FoxP3 mRNA expression
were equivalent in BALB/c, CBA/J, and MRL/lpr
CD4⫹,CD25⫹ LN T cells. The expression levels of
FoxP3 protein as determined by intracellular staining
(Figure 1C) and the perichromatin localization visualized by electron microscopy with immunogold labeling
(not shown) of CD4⫹,CD25⫹ T cells from BALB/c,
CBA/J, and MRL/lpr mice were similar. Thus, the
proportion of CD4⫹,CD25⫹,CD62L⫹,FoxP3⫹ T cells
in the peripheral CD4⫹ T cell population, as well as the
expression of FoxP3 in LN CD4⫹,CD25⫹ T cells, are
similar in MRL/lpr lupus mice and normal mice.
Capacity of MRL/lpr CD4ⴙ,CD25ⴙ T cells to
suppress effector T cells in response to polyclonal stimulation. The capacity of MRL/lpr and H2-matched
CBA/J CD4⫹,CD25⫹ T cells to inhibit effector T cells
in response to polyclonal activation was tested by incubating CD4⫹,CD25⫺ effector T cells, stimulated with
both anti-CD3 mAb and haplotype-matched APCs, with
CD4⫹,CD25⫹ T cells (ratio 1:1). CD4⫹,CD25⫹ T cells
purified from MRL/lpr mice displayed an anergic phenotype (no proliferation, no IL-2, and very low levels of
IFN␥ secretion) when stimulated by anti-CD3 mAb and
autologous APCs (Figure 2). In these conditions of
activation, we observed a reduced capacity of MRL/lpr
Treg cells to inhibit the proliferation of syngeneic effector T cells, compared with syngeneic cocultures with
CBA/J Treg cells and effector T cells (65% versus 81%;
P ⫽ 0.003) (Figure 2A). Dose-response curves using
graded numbers of CD4⫹,CD25⫹ T cells also revealed
the more potent suppressive activity of CBA/J Treg cells
(Figure 2A). The ability of CD4⫹,CD25⫹ T cells to
reduce IL-2 secretion by effector T cells was apparently
equivalent in both groups of mice. However, this result
should be interpreted with caution, since stimulated
MRL/lpr effector T cells are known to be poor IL-2
producers (34,35).
Finally, compared with syngeneic CBA/J cocultures, we found a statistically significant lower efficacy of
MRL/lpr Treg cells to inhibit IFN␥ secretion by syngeneic CD4⫹,CD25⫺ T cells (51% versus 94%; P ⬍ 0.001)
(Figure 2B). It should be noted that in some experiments, measurable levels of IFN␥ were produced by
MRL/lpr Treg cells. However, this low secretion cannot
account for the raised amounts measured in MRL/lpr
coculture conditions, and it is not related to contamination with activated T cells that secrete IFN␥, since 94%
of purified CD4⫹,CD25⫹ T cells used in these experiments are FoxP3⫹ cells (i.e., Treg cells).
Cocultures were also performed with cells isolated from MRL/Mp mice. Only a weak T cell activation
was generated, which precluded our calculating the level
of suppression with statistical certainty.
IMPAIRMENT OF TREG CELLS IN MRL/lpr MICE
1755
Figure 2. Reduced capacity of MRL/lpr Treg cells, which have an anergic phenotype, to suppress proliferation and interferon-␥ (IFN␥) secretion
by effector T cells. CD4⫹,CD25⫺ T cells or CD4⫹,CD25⫹ T cells or the 2 populations mixed in a 1:1 ratio, purified from lymph nodes of four
10-week-old CBA/J mice or four 10-week-old MRL/lpr mice, were cultured in the absence or presence of anti-CD3 monoclonal antibodies and
syngeneic antigen-presenting cells (APCs). A, Proliferative responses were measured on day 3. Results are expressed as the mean and SD cpm of
triplicate cultures in 4 independent experiments, with the percentages of suppression of proliferation indicated above the bars, or as the mean ⫾
SD percentage of inhibition of the proliferative response in relation to the CD4⫹,CD25⫹ T cell:CD4⫹,CD25⫺ T cell ratio. The average
3
H-thymidine incorporation in the absence of stimulation was 50 cpm. B, Interleukin-2 (IL-2) and IFN␥ secretion were measured by double-sandwich
enzyme-linked immunosorbent assay in 48-hour supernatants. Values are the mean and SD, with the percentages of suppression of IL-2 and IFN␥
secretion indicated above the bars.
Altered functionality of each partner involved in
peripheral regulation (Treg cells, effector T cells, and
APCs) in MRL/lpr lupus mice. To determine whether
this defective function described above was attributable
to MRL/lpr Treg cells or to decreased sensitivity to
suppression of MRL/lpr CD4⫹,CD25⫺ effector T cells,
we performed crossover experiments (Figure 3). Compared with CBA/J Treg cells, when MRL/lpr Treg cells
were used with CBA/J effector T cells and CBA/J APCs,
there was less inhibition of both proliferation (63%
versus 81%; P ⬍ 0.001) and IFN␥ secretion (70% versus
94%; P ⫽ 0.003) (Figure 3, open bars), suggesting that
the regulatory functions of Treg cells are affected in
MRL/lpr mice. However, cocultures of CBA/J Treg cells
with effector T cells and APCs from MRL/lpr mice
(Figure 3, solid bars) revealed that MRL/lpr
CD4⫹,CD25⫺ T cells were strongly refractory to suppression (inhibition of proliferation 51% versus 81%;
P ⬍ 0.001) (inhibition of IFN␥ secretion 59% versus
94%; P ⫽ 0.001). These data demonstrate clearly that a
major defect of MRL/lpr effector CD4⫹ T cells is their
inability to be fully suppressed by Treg cells (regardless
1756
PARIETTI ET AL
Figure 3. Altered functionality of each partner (Treg cells, effector T cells, and antigen-presenting cells [APCs]) involved in peripheral regulation.
CD4⫹,CD25⫹ Treg cells were tested for their ability to suppress proliferation and interferon-␥ (IFN␥) production of CD4⫹,CD25⫺ effector T cells
stimulated with mitomycin C–treated T cell–depleted APCs and anti-CD3 monoclonal antibodies. The efficiency of CD4⫹,CD25⫹ Treg cells from
five 10-week-old CBA/J (CBA) mice or five 10-week-old MRL/lpr (lpr) mice was evaluated by testing CD4⫹,CD25⫺ T cells purified from CBA/J
or MRL/lpr mice stimulated by either MRL/lpr APCs or CBA/J APCs. Top left, Proliferation was assessed on day 3, and results are expressed as
the mean and SD cpm from triplicate cultures in 4 independent experiments. Top right, The mean and SD percentage of inhibition of proliferation
was calculated. Bottom left, IFN␥ levels were measured by double-sandwich enzyme-linked immunosorbent assay in 48-hour supernatants, and
results are expressed as the mean and SD IFN␥ concentration from triplicate cultures in 4 independent experiments. Bottom right, The mean and
SD percentage of inhibition of IFN␥ secretion was calculated. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001.
of the origin of these cells, from lupus or normal mice).
The reduced sensitivity of MRL/lpr CD4⫹,CD25⫺ T
cells to suppression was not due either to an increased
proliferation of these CD4⫹,CD25⫺ T cells or to defective apoptosis of effector cells (data not shown), since it
could be envisaged in MRL/lpr mice that are characterized by a Fas/FasL pathway deficiency.
In vitro, Treg cells exert their activity when
stimulated by anti-CD3 mAb mimicking TCR signaling
and a secondary signal that can be provided by APCs. To
investigate the potential role of MRL/lpr APCs in the
defective activation of Treg cells, we performed crossover experiments in which APCs from MRL/lpr and
CBA/J mice were cocultured with effectors and Treg
cells from both strains of mice. Surprisingly, adding
APCs from MRL/lpr mice to CD4⫹,CD25⫹ and
CD4⫹,CD25⫺ T cells from CBA/J mice was sufficient
to reduce the capacity of Treg cells to suppress effector
T cells (inhibition of proliferation and IFN␥ secretion
were decreased from 81% to 59% and from 94% to
59%, respectively; both P ⫽ 0.005) (Figure 3). However,
CBA/J APCs cocultured with MRL/lpr T cells were not
able to restore suppressive functions (inhibition of proliferation 37% and inhibition of IFN␥ secretion 59%).
Taken together, these data strongly suggest that in
MRL/lpr mice, APCs, CD4⫹,CD25⫹ T cells, and
CD4⫹,CD25⫺ T cells each display abnormal functions.
We then performed crossover experiments using
IMPAIRMENT OF TREG CELLS IN MRL/lpr MICE
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Figure 4. Reduced susceptibility to suppression in effector
CD4⫹,CD25⫺ T cells from MRL/lpr (lpr) mice. The suppression of
interferon-␥ (IFN␥) production by effector T cells was analyzed by
coculturing CD4⫹,CD25⫹ Treg cells and CD4⫹,CD25⫺ effector T
cells stimulated with mitomycin C–treated T cell–depleted antigenpresenting cells (APCs) and anti-CD3 monoclonal antibodies. IFN␥
levels were measured by double-sandwich enzyme-linked immunosorbent assay in 48-hour supernatants, and results are expressed as the
mean and SD percentage of inhibition. ⴱⴱⴱ ⫽ P ⬍ 0.001. CBA ⫽
CBA/J; Mp ⫽ MRL/Mp.
cells from MRL/Mp mice. As described above, the
hyporesponsive state of MRL/Mp CD4⫹,CD25⫺ effector T cells to polyclonal stimulation did not allow us to
measure any significant suppression. Thus, we focused
on the major defect we detected in MRL/lpr mice, that
is, the inability of MRL/lpr effector T cells to be fully
suppressed. We evaluated the inhibition of IFN␥ secretion by MRL/lpr CD4⫹,CD25⫺ T cells cocultured with
Treg cells and APCs from normal CBA/J or MRL/Mp
mice. As shown in Figure 4, MRL/lpr effector T cells
were strongly refractory to suppression, regardless of the
origin of Treg cells and APCs. Results obtained with
Treg cells and APCs from either CBA/J or MRL/Mp
mice were quite similar (66% and 61% inhibition,
respectively, compared with 95% inhibition in CBA/J
cocultures; both P ⬍ 0.001), demonstrating that an
altered interaction between cells of 2 different strains
(i.e., CBA/J and MRL/lpr mice) cannot explain the
refractory behavior of MRL/lpr T cells to suppression.
Diminished expression of CTLA-4 and CD80/
CD86 on MRL/lpr cells may compromise the crosstalk
between the cellular partners involved in suppressive
mechanisms. Proposed mechanisms for the suppressive
activity of Treg cells involve the interaction of CTLA-4
with CD80/CD86 molecules expressed not only on
Figure 5. Reduced expression of CTLA-4, CD80, and CD86 molecules implicated in suppressive mechanisms. A, Intracellular expression of CTLA-4 by Treg cells was assessed by flow cytometry following
24-hour activation by anti-CD3 monoclonal antibodies (mAb) and
mitomycin C–treated T cell–depleted antigen-presenting cells (APCs)
pooled from three 10-week-old CBA/J (CBA) mice or three 10-weekold MRL/lpr (lpr) mice. Cells were stained with allophycocyaninlabeled anti-CD4, fluorescein isothiocyanate–labeled anti-CD25, and
phycoerythrin-labeled anti–CTLA-4. Representative expression of
CTLA-4 by Treg cells is shown as fluorescence-activated cell sorting
histograms (shaded areas correspond to staining with isotype control
antibodies; solid lines correspond to CTLA-4 staining). The mean and
SD mean fluorescence intensity (MFI) levels are represented as a
histogram. B, Expression of CD80 and CD86 was determined on
CBA/J, MRL/Mp (Mp), and MRL/lpr effector CD4⫹,CD25⫺ T cells
after 24 hours of activation by anti-CD3 mAb and syngeneic mitomycin
C–treated T cell–depleted APCs. The histograms represent CD80 and
CD86 expression on activated effector T cells (Eff) identified as
CD4⫹,B220⫹ T cells. The activation was also performed in crossover
experiments (hatched histograms), and the mean and SD MFI levels of
CD80 and CD86 for each condition are shown. C, Expression of CD80
and CD86 molecules was analyzed on CBA/J and MRL/lpr mitomycin
C–treated T cell–depleted APCs cultured for 24 hours with autologous
effector CD4⫹,CD25⫺ T cells and anti-CD3 mAb. ⴱ ⫽ P ⱕ 0.05; ⴱⴱ
⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001.
1758
APCs, but also on recently activated T cells (36). Since
Treg cells, APCs, and activated/effector T cells from
MRL/lpr mice seem to present defective functions, we
analyzed the expression of these 2 key markers on the
respective cell populations. Treg cells from CBA/J and
MRL/lpr mice were compared by intracellular staining
for CTLA-4 expression (Figure 5A). Since the minimum
number of Treg cells (⬃106) required to perform experiments under all conditions did not allow us to analyze
each mouse individually, this experiment was done using
Treg cells pooled from 3 mice. When stimulated by
CBA/J APCs, the level of CTLA-4 expression on MRL/
lpr Treg cells was reduced compared with the level
observed on normal CBA/J Treg cells (mean fluorescence intensity [MFI] 21 versus 34; P ⫽ 0.05) (Figure
5A). Moreover, when stimulated with MRL/lpr APCs,
this diminution of CTLA-4 expression was more pronounced on MRL/lpr Treg cells (MFI 15; P ⫽ 0.05 versus
normal CBA/J Treg cells) (Figure 5A), suggesting that in
addition to an intrinsic defect in Treg cells, MRL/lpr
APCs are not able to efficiently induce the expression of
CTLA-4 on Treg cells.
We next analyzed the expression of CD80/CD86
molecules on activated/effector T cells, and we observed
that CD80 and CD86 expression levels following 24
hours of activation were lower on activated T cells from
MRL/lpr lupus mice than on activated T cells from
CBA/J and MRL/Mp mice (for CD80, MFI 101 versus
226 and 260, respectively; for CD86, MFI 90 versus 400
and 450, respectively) (Figure 5B). Moreover, the expression of CD80 and CD86 was dramatically decreased
on effector T cells from CBA/J mice activated by
MRL/lpr APCs (for CD80, MFI from 226 to 93; for
CD86, MFI from 400 to 90) (Figure 5B). When effector
T cells from lupus mice were activated with CBA/J
APCs, the expression levels of CD80 and CD86 were
enhanced but did not reach those obtained on CBA/J
effector T cells (for CD80, MFI 150; for CD86, MFI
220). Our results suggest that defective expression of
CTLA-4 on Treg cells and of CD80 and CD86 on
effector T cells depends on the origin of APCs used to
stimulate the culture. Furthermore, the analysis of CD80
and CD86 expression on APCs (almost exclusively B
cells after 24 hours of activation) revealed a lowered
expression on MRL/lpr APCs, with the existence of a
subpopulation clearly negative for CD86 compared with
CBA/J APCs (Figure 5C) and compared with MRL/Mp
APCs (data not shown).
The altered functionality of MRL/lpr Treg cells is
due to an overproduction of IL-6 by MRL/lpr APCs. We
examined the characteristics of APCs (which correspond
PARIETTI ET AL
Figure 6. Involvement of interleukin-6 (IL-6) secreted by MRL/lpr
antigen-presenting cells (APCs) in defective Treg cell–mediated suppression. A, T cell–depleted APCs from 8-week-old CBA/J (CBA) mice,
11-week-old MRL/Mp mice, 5-week-old MRL/lpr mice (prediseased), and
10-week-old MRL/lpr mice were stimulated with increasing concentrations of lipopolysaccharide (LPS). IL-6 levels in 24-hour supernatants
were quantified by enzyme-linked immunosorbent assay (ELISA). Values
are the mean ⫾ SD. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, versus CBA/J,
MRL/Mp, and prediseased MRL/lpr mice. B, CBA/J effector T cells were
activated with anti-CD3 monoclonal antibodies (mAb) and mitomycin
C–treated T cell–depleted APCs from CBA/J or MRL/lpr mice. Neutralizing anti–IL-6 mAb were added in control culture. IL-6 levels in 48-hour
supernatants were quantified by ELISA. Values are the mean and SD. C,
CBA/J CD4⫹,CD25⫺ T cells alone or mixed in a 1:1 ratio with CBA/J
CD4⫹,CD25⫹ T cells were stimulated with anti-CD3 mAb and mitomycin C–treated T cell–depleted APCs from CBA/J or MRL/lpr mice.
Anti–IL-6 mAb was added as indicated. Interferon-␥ (IFN␥) secretion in
48-hour supernatants was determined by ELISA. Values are the mean
and SD. Percentages of inhibition are indicated. ND ⫽ not determined.
to a T cell–depleted splenocyte fraction) used in our
assays. Since IL-6 produced by Toll-like receptor 4
(TLR-4)–activated dendritic cells was described as a
IMPAIRMENT OF TREG CELLS IN MRL/lpr MICE
soluble factor able to block the suppressive effect of
Treg cells (37), we investigated the involvement of this
cytokine in our system. We compared the IL-6 secretion
levels generated by LPS-activated APCs (mainly B cells
in our cell extract) from CBA/J, MRL/Mp, and MRL/lpr
mice. The levels of IL-6 secreted by LPS-activated APCs
from CBA/J, MRL/Mp, and prediseased MRL/lpr mice
were equivalent (Figure 6A). In contrast, APCs from
10-week-old MRL/lpr mice produced significantly higher
levels of IL-6 when stimulated with LPS. The concentration of LPS required to induce IL-6 secretion by
APCs from 10-week-old MRL/lpr mice was ⬃50-fold
lower than the concentration of LPS necessary to stimulate APCs from CBA/J, MRL/Mp, or young MRL/lpr
mice (Figure 6A). Raised amounts of IL-6 were also
detected when MRL/lpr APCs were cultured with CBA/J
effector T cells (390 pg/ml versus 80 pg/ml, when CBA/J
APCs were in the cocultures; P ⫽ 0.004). This production was not detected in the presence of neutralizing
anti–IL-6 mAb (Figure 6B). Interestingly, we observed that
defective suppression in MRL/lpr coculture was highly
correlated with IL-6 secretion by APCs (data not shown).
To further study the involvement of IL-6 in
MRL/lpr Treg cell functionality, we analyzed the inhibition of IFN␥ secretion in cocultures of APCs, effector T
cells, and Treg cells in the presence or absence of
neutralizing anti–IL-6 mAb. In cocultures with APCs
and effector T cells isolated from CBA/J mice, we
confirmed that IFN␥ secretion was dramatically inhibited (94%) by CBA/J Treg cells (Figure 6C). As described above, the production of IFN␥ by effector T cells
from CBA/J mice stimulated with MRL/lpr APCs was
only partially inhibited (67%) by CBA/J Treg cells.
Interestingly, the addition of neutralizing anti–IL-6 mAb
was sufficient to restore the functionality of CBA/J Treg
cells, since the IFN␥ secretion was almost completely
(90%) inhibited (Figure 6C). No effect on the IFN␥
secretion was observed when anti–IL-6 mAb was added
to cultures without Treg cells, indicating that the anti–
IL-6 mAb exerts its activity on Treg cell functions and
not on effector T cells.
DISCUSSION
In addition to central control mechanisms such as
clonal deletion and anergy, the maintenance of tolerance in the periphery is supported by Treg cells. Since
thymic tolerance appears intact in MRL/lpr lupus-prone
mice (38), it has been postulated that in lupus mice, the
pathogenic autoantibody production is driven by activated T cells, which presumably bypass normal tolerance
mechanisms in the periphery. The regulation of these
1759
cells by Treg cells has been evaluated in different
situations but has never been explored in MRL/lpr mice.
In the present study, we asked whether in this lupus
strain, Treg cells could display numerical and/or functional alteration. We first analyzed the percentage of
Treg cells in the periphery and demonstrated that
MRL/lpr lupus mice, in which a fatal immune complex
glomerulonephritis develops, are not deficient in
CD4⫹,CD25⫹,FoxP3⫹ Treg cells.
We next investigated whether these Treg cells
display normal functions. In good concordance with the
results obtained in MRL/Mp mice (32), we demonstrated that in MRL/lpr mice, CD4⫹,CD25⫺ T cells are
also substantially less susceptible to T cell suppression. It
was thus critical to determine in MRL/lpr mice whether
the decrease in the Treg cell function was due to an
intrinsic defect in the CD4⫹,CD25⫹ T cell population
or to the inability of responder CD4⫹,CD25⫺ T cells
themselves to be suppressed. Using crossover experiments and polyclonal stimulation, we clearly demonstrated that, in fact, both cell subsets are affected in
MRL/lpr mice. Indeed, we also observed that MRL/lpr
CD4⫹,CD25⫹ Treg cells have a reduced capacity to
inhibit the proliferation of effector T cells and IFN␥
secretion.
The most relevant result to be considered is the
fact that when cultured in syngeneic conditions (mimicking the function they should maintain in vivo), MRL/
lpr Treg cells are not able to control the activation of
effector T cells. Our crossover experiments revealed that
MRL/lpr Treg cell functions are only slightly altered
when these cells are cultured in the presence of CBA/J
APCs and CBA/J effector T cells, suggesting that in
MRL/lpr mice, the major defect resides in effector T
cells and APCs. This was confirmed by culturing regulatory and effector T cells from CBA/J mice with
MRL/lpr APCs. In these conditions, the capacity of
CBA/J Treg cells to suppress proliferation (and IFN␥
secretion) of effector T cells was significantly affected,
indicating that Treg cells were not fully activated. It was
recently highlighted that the conditions of activation
used to stimulate Treg cells and effector T cells are
crucial to evaluate Treg cell functionality (39). The
secretion, by APCs, of soluble factors preventing the
suppressive properties of CD4⫹,CD25⫹ T cells in vitro
underlines the fact that any study analyzing Treg cell
functionality should be performed under conditions of
activation that use autologous APCs. Our study is therefore likely to be more informative than the study performed in the MRL/Mp model, in which effector T cells
1760
and Treg cells were stimulated with anti-CD3 and
anti-CD28 antibody–coated beads (32).
Although the suppressive mechanisms mediated
by Treg cells are poorly understood, a major role
appears to be played by cell–cell interactions rather than
by soluble molecules (40,41). The suppressive activity of
Treg cells depends on signaling via CTLA-4 and its
interaction with CD80/CD86 molecules. In the present
study, we observed that the levels of CD80 and CD86
molecules expressed after activation on both effector T
cells and APCs from MRL/lpr mice was not optimal. In
line with this, MRL/lpr Treg cells express lower levels of
CTLA-4 than CBA/J Treg cells. The impaired expression of these surface molecules could explain the inefficient regulatory function of MRL/lpr Treg cells.
It has been demonstrated that soluble factors,
and especially IL-6 produced by dendritic cells stimulated with TLR-4 and TLR-9 ligands, can block Treg
cell–mediated suppression (37). In a triple Sle–congenic
lupus-prone mouse, it was recently shown that overproduction of IL-6 by dendritic cells was involved in the
blockade of Treg cell activity (42). We also observed that
when exposed to LPS, MRL/lpr APCs secrete higher
IL-6 levels than CBA/J APCs. Moreover, using a neutralizing anti–IL-6 mAb, we clearly showed that this
overproduction of IL-6 may be responsible for the
altered functionality of MRL/lpr Treg cells. These results are particularly interesting with regard to the
involvement of TLRs in lupus pathogenesis. Indeed, a
persistent activation of any of the TLR pathways could
lead to an aberrant production and/or expression of
molecules responsible for the inhibition of Treg cell
functions in MRL/lpr lupus mice.
Soluble factors other than IL-6 could also be
involved in the impaired suppressive function of Treg
cells. It was recently proposed that excessive TNF␣
production and diminished IL-2 production may contribute to this defect (22). Since it is known that T cells
from MRL/lpr mice generate low levels of IL-2 upon in
vitro stimulation with concanavalin A (34,35), it is
possible that the defective function of MRL/lpr Treg
cells is due to inappropriate, suboptimal amounts of IL-2
secreted by effector T cells. This hypothesis is supported
by our results, which show the inability of CBA/J and
MRL/lpr Treg cells to suppress IFN␥ production when
cultured (i.e., stimulated) with IL-2–defective MRL/lpr
effector T cells. This point is of particular interest, since
a defect in IL-2 production was also described in patients with SLE (43,44).
In conclusion, our data show that in MRL/lpr
mice, major defects in the regulation process concern
PARIETTI ET AL
both effector T cells (less susceptible to suppression)
and APCs. Interestingly, impaired expression of CD80/
CD86 molecules on APCs, as well as the excessive
production of IL-6 described in this study, are also
common features of patients with lupus (45–47). This
highlights the fact that MRL/lpr mice can be a useful
model to develop immunomodulatory strategies through
the manipulation of Treg cells.
AUTHOR CONTRIBUTIONS
Dr. Muller had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Parietti, Monneaux, Muller.
Acquisition of data. Parietti, Monneaux, Décossas.
Analysis and interpretation of data. Parietti, Monneaux, Muller.
Manuscript preparation. Parietti, Monneaux, Muller.
Statistical analysis. Parietti, Monneaux.
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