Function of CD4+CD25+ Treg cells in MRLlpr mice is compromised by intrinsic defects in antigen-presenting cells and effector T cells.код для вставкиСкачать
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. 1751 1752 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 1757 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. REFERENCES 1. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor ␣-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151–64. 2. Von Boehmer H. Mechanisms of suppression by suppressor T cells. Nat Immunol 2005;6:338–44. 3. Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25⫹CD4⫹ regulatory cells that control intestinal inflammation. J Exp Med 2000;192:295–302. 4. McHugh RS, Whitters MJ, Piccirillo CA, Young DA, Shevach EM, Collins M, et al. CD4⫹CD25⫹ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoidinduced TNF receptor. Immunity 2002;16:311–23. 5. Marski M, Kandula S, Turner JR, Abraham C. CD18 is required for optimal development and function of CD4⫹CD25⫹ T regulatory cells. J Immunol 2005;175:7889–97. 6. Thornton AM, Piccirillo CA, Shevach EM. Activation requirements for the induction of CD4⫹CD25⫹ T cell suppressor function. Eur J Immunol 2004;34:366–76. 7. Houot R, Perrot I, Garcia E, Durand I, Lebecque S. Human CD4⫹CD25high regulatory T cells modulate myeloid but not plasmacytoid dendritic cells activation. J Immunol 2006;176: 5293–8. 8. Zhao DM, Thornton AM, DiPaolo RJ, Shevach EM. Activated CD4⫹CD25⫹ T cells selectively kill B lymphocytes. Blood 2006; 107:3925–32. 9. Lim HW, Hillsamer P, Banham AH, Kim CH. Cutting edge: direct suppression of B cells by CD4⫹CD25⫹ regulatory T cells. J Immunol 2005;175:4180–3. 10. Morgan ME, Sutmuller RP, Witteveen HJ, van Duivenvoorde LM, Zanelli E, Melief CJ, et al. CD25⫹ cell depletion hastens the onset of severe disease in collagen-induced arthritis. Arthritis Rheum 2003;48:1452–60. 11. Seo SJ, Fields ML, Buckler JL, Reed AJ, Mandik-Nayak L, Nish SA, et al. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 2002; 16:535–46. 12. Balandina A, Lecart S, Dartevelle P, Saoudi A, Berrih-Aknin S. Functional defect of regulatory CD4⫹CD25⫹ T cells in the thymus IMPAIRMENT OF TREG CELLS IN MRL/lpr MICE 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. of patients with autoimmune myasthenia gravis. Blood 2005;105: 735–41. Mqadmi A, Zheng X, Yazdanbakhsh K. CD4⫹CD25⫹ regulatory T cells control induction of autoimmune hemolytic anemia. Blood 2005;105:3746–8. Boyer O, Saadoun D, Abriol J, Dodille M, Piette JC, Cacoub P, et al. CD4⫹CD25⫹ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood 2004;103: 3428–30. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of functional suppression by CD4⫹CD25⫹ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004;199:971–9. Ehrenstein MR, Evans JG, Singh A, Moore S, Warnes G, Isenberg DA, et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF␣ therapy. J Exp Med 2004;200:277–85. Kriegel MA, Lohmann T, Gabler C, Blank N, Kalden JR, Lorenz HM. Defective suppressor function of human CD4⫹ CD25⫹ regulatory T cells in autoimmune polyglandular syndrome type II. J Exp Med 2004;199:1285–91. Van Amelsfort JM, Jacobs KM, Bijlsma JW, Lafeber FP, Taams LS. CD4⫹CD25⫹ regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum 2004;50: 2775–85. Sugiyama H, Gyulai R, Toichi E, Garaczi E, Shimada S, Stevens SR, et al. Dysfunctional blood and target tissue CD4⫹CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation. J Immunol 2005;174: 164–73. Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TI. Defective suppressor function in CD4⫹CD25⫹ T-cells from patients with type 1 diabetes. Diabetes 2005;54:92–9. Alvarado-Sanchez B, Hernandez-Castro B, Portales-Perez D, Baranda L, Layseca-Espinosa E, Abud-Mendoza C, et al. Regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun 2006;27:110–8. Valencia X, Yarboro C, Illei G, Lipsky PE. Deficient CD4⫹ CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 2007;178:2579–88. Miyara M, Amoura Z, Parizot C, Badoual C, Dorgham K, Trad S, et al. Global natural regulatory T cell depletion in active systemic lupus erythematosus. J Immunol 2005;175:8392–400. Cao D, Malmstrom V, Baecher-Allan C, Hafler D, Klareskog L, Trollmo C. Isolation and functional characterization of regulatory CD25brightCD4⫹ T cells from the target organ of patients with rheumatoid arthritis. Eur J Immunol 2003;33:215–23. Gottenberg JE, Lavie F, Abbed K, Gasnault J, Le Nevot E, Delfraissy JF, et al. CD4 CD25high regulatory T cells are not impaired in patients with primary Sjögren’s syndrome. J Autoimmun 2005;24:235–42. Liu MF, Wang CR, Fung LL, Wu CR. Decreased CD4⫹CD25⫹ T cells in peripheral blood of patients with systemic lupus erythematosus. Scand J Immunol 2004;59:198–202. Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun 2003;21:273–6. Lee JH, Wang LC, Lin YT, Yang YH, Lin DT, Chiang BL. Inverse correlation between CD4⫹ regulatory T-cell population and autoantibody levels in paediatric patients with systemic lupus erythematosus. Immunology 2006;117:280–6. Suarez A, Lopez P, Gomez J, Gutierrez C. Enrichment of 1761 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. CD4⫹CD25high T cell population in SLE patients treated with glucocorticoids. Ann Rheum Dis 2006;35:22–5. Wu HY, Staines NA. A deficiency of CD4⫹CD25⫹ T cells permits the development of spontaneous lupus-like disease in mice, and can be reversed by induction of mucosal tolerance to histone peptide autoantigen. Lupus 2004;13:192–200. Scalapino KJ, Tang Q, Bluestone JA, Bonyhadi ML, Daikh DI. Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells. J Immunol 2006;177:1451–9. Monk CR, Spachidou M, Rovis F, Leung E, Botto M, Lechler RI, et al. MRL/Mp CD4⫹,CD25⫺ T cells show reduced sensitivity to suppression by CD4⫹,CD25⫹ regulatory T cells in vitro: a novel defect of T cell regulation in systemic lupus erythematosus. Arthritis Rheum 2005;52:1180–4. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299: 1057–61. Wofsy D, Murphy ED, Roths JB, Dauphinee MJ, Kipper SB, Talal N. Deficient interleukin 2 activity in MRL/Mp and C57BL/6J mice bearing the lpr gene. J Exp Med 1981;154:1671–80. Altman A, Theofilopoulos AN, Weiner R, Katz DH, Dixon FJ. Analysis of T cell function in autoimmune murine strains: defects in production and responsiveness to interleukin 2. J Exp Med 1981;154:791–808. Taylor PA, Lees CJ, Fournier S, Allison JP, Sharpe AH, Blazar BR. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J Immunol 2004; 172:34–9. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4⫹ CD25⫹ T cell-mediated suppression by dendritic cells. Science 2003;299:1033–6. Fatenejad S, Peng SL, Disorbo O, Craft J. Central T cell tolerance in lupus-prone mice: influence of autoimmune background and the lpr mutation. J Immunol 1998;161:6427–32. Mudd PA, Teague BN, Farris AD. Regulatory T cells and systemic lupus erythematosus. Scand J Immunol 2006;64:211–8. Shevach EM. CD4⫹CD25⫹ suppressor T cells: more questions than answers. Nat Rev Immunol 2002;2:389–400. Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat Immunol 2001;2:816–22. Wan S, Xia C, Morel L. IL-6 produced by dendritic cells from lupus-prone mice inhibits CD4⫹CD25⫹ T cell regulatory functions. J Immunol 2007;178:271–9. Linker-Israeli M, Bakke AC, Kitridou RC, Gendler S, Gillis S, Horwitz DA. Defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus (SLE). J Immunol 1983;130:2651–5. Alcocer-Varela J, Alarcon-Segovia D. Decreased production of and response to interleukin-2 by cultured lymphocytes from patients with systemic lupus erythematosus. J Clin Invest 1982;69: 1388–92. Garcia-Cozar FJ, Molina IJ, Cuadrado MJ, Marubayashi M, Pena J, Santamaria M. Defective B7 expression on antigen-presenting cells underlying T cell activation abnormalities in systemic lupus erythematosus (SLE) patients. Clin Exp Immunol 1996;104:72–9. Liu MF, Li JS, Weng TH, Lei HY. Differential expression and modulation of costimulatory molecules CD80 and CD86 on monocytes from patients with systemic lupus erythematosus. Scand J Immunol 1999;49:82–7. Tackey E, Lipsky PE, Illei GG. Rationale for interleukin-6 blockade in systemic lupus erythematosus. Lupus 2004;13:339–43.