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Proc Natl Acad Sci U S A. Jan 5, 2010; 107(1): 204–209.
Published online Dec 29, 2009. doi:  10.1073/pnas.0903158107
PMCID: PMC2806746
Immunology

Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus

Abstract

The origins and consequences of a regulatory T cell (Treg) disorder in systemic lupus erythematosus (SLE) are poorly understood. In the (NZBxNZW) F1 mouse model of lupus, we found that CD4+Foxp3+ Treg failed to maintain a competitive pool size in the peripheral lymphoid organs resulting in a progressive homeostatic imbalance of CD4+Foxp3+ Treg and CD4+Foxp3 conventional T cells (Tcon). In addition, Treg acquired phenotypic changes that are reminiscent of IL-2 deficiency concomitantly to a progressive decline in IL-2-producing Tcon and an increase in activated, IFN-γ-producing effector Tcon. Nonetheless, Treg from lupus-prone mice were functionally intact and capable to influence the course of disease. Systemic reduction of IL-2 levels early in disease promoted Tcon hyperactivity, induced the imbalance of Treg and effector Tcon, and strongly accelerated disease progression. In contrast, administration of IL-2 partially restored the balance of Treg and effector Tcon by promoting the homeostatic proliferation of endogenous Treg and impeded the progression of established disease. Thus, an acquired and self-amplifying disruption of the Treg-IL-2 axis contributed essentially to Tcon hyperactivity and the development of murine lupus. The reversibility of this homeostatic Treg disorder provides promising approaches for the treatment of SLE.

Keywords: homeostasis, immunotherapy, interleukin-2, SLE, autoimmunity

Regulatory CD4+ T cells (Treg) that express the transcription factor Foxp3 are crucial for the maintenance of immunological tolerance to self (1, 2). Predominantly derived from a distinct T cell subpopulation in the thymus, CD4+Foxp3+ Treg principally recognize self-antigens and are required to control the expansion of self-reactive T cells in the peripheral lymphoid organs (2, 3). In view of that, there is increasing evidence that numeric or functional Treg deficiencies are associated with particular autoimmune diseases, suggesting a contribution of a Treg dysfunction to disease development (4).

The cytokine IL-2 was initially identified as a potent T cell growth factor (5). However, more recent data strongly indicate that IL-2 is essential for immune tolerance (5). Accordingly, mice deficient in IL-2 or IL-2 receptor components, including CD25, succumb to a rapidly progressing autoimmune disease that is caused by an uncontrolled activation of CD4+ T cells and B cells (68). The fundamental function of IL-2 in Treg biology was recently highlighted with the demonstration that IL-2 was critically required for the homeostatic maintenance of Treg in the peripheral lymphoid organs (911). Other studies have also suggested a requirement of IL-2 for the suppressive function and the thymic development of Treg (12, 13). Therefore, disturbances in the Treg-IL-2 axis can result in autoimmunity or contribute to the development of immune-mediated diseases.

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease with complex genetics and unknown etiology. It is characterized by a breakdown of tolerance to ubiquitous nuclear antigens, including double-stranded DNA (ds-DNA), that results in an uncontrolled activation of self-reactive B and T cells and consequent multiorgan inflammation, most importantly nephritis (14, 15). Despite a few contradictory reports, most studies have found a low prevalence of CD4+CD25+ T cells in SLE patients and murine SLE models, suggesting a disturbed maintenance of the Treg pool size (1618). Nonetheless, the interpretation of most of these studies may be restricted because of the common identification of Treg by CD25, a surface marker that is also expressed by activated conventional CD4+ T cells (Tcon) and absent in a large proportion of Treg.

Impaired IL-2 production by T cells has also been attributed a critical role in murine and human SLE (19, 20). However, it remains unclear whether a shortage of IL-2 causes abnormalities of the Treg population in lupus-prone individuals and how such a disturbance is causally linked to the pathogenesis of this disease.

To address these fundamental questions, we explored the origins and consequences of abnormalities in the CD4+Foxp3+ Treg pool during the course of SLE progression using (NZBxNZW) F1 lupus mice, a spontaneous autoimmune model that displays many features of human SLE, including fatal nephritis (21). We found that the lupus-prone mice failed to sustain a competitive number of CD4+Foxp3+ Treg in the peripheral lymphoid organs because of an acquired deficiency of IL-2 and IL-2-producing CD4+ T cells. This homeostatic impairment of Treg boosted Tcon hyperactivity, resulting in a progressive imbalance of Treg and effector Tcon, and promoted the progression of disease. Furthermore, we showed that compensation of the IL-2 deficiency in lupus mice by treatment with rIL-2 impeded the progression of established disease most likely by re-establishing the homeostatic balance of Treg and effector Tcon, indicating the reversibility of this acquired Treg disorder.

Results

Progressive Homeostatic Imbalance of Treg and Tcon.

The percentage and absolute numbers of CD4+Foxp3+ Treg were evaluated in different organs and at different time points during the development of disease in (NZBxNZW) F1 mice by flow cytometry. A comparison between young animals (young), animals at the onset of disease (onset), and old, diseased animals (diseased) showed a progressive deficiency in the number of Treg in the lymph nodes and the peripheral blood. (Fig. 1 A and B). This deficiency of Treg was contrasted to progressive increases in the percentage and absolute numbers of Treg in the spleens and of CD4 single positive (SP) Treg in the thymi (Fig. 1 A and B).

Fig. 1.
Progressive homeostatic imbalance of Treg and Tcon. (A and B) Average percentage of Foxp3+ cells among CD4+ and among CD4 SP T cells (A) and absolute numbers (B) of CD4+Foxp3+ T cells in spleens (SN), lymph nodes (LN), peripheral blood (PB), and of CD4 ...

Next, we studied the in vivo proliferation of CD4+Foxp3+ Treg and CD4+Foxp3 Tcon by BrdU incorporation. The proliferation rates of Treg and Tcon in lymphoid organs from young (NZBxNZW) F1 mice were not different from those in aged-matched BALB/c mice (Fig. S1A). However, this changed in lupus mice at the onset of disease: the proliferation rate of Treg was now lower in spleens and lymph nodes (Fig. S1A) compared to aged-matched BALB/c mice, whereas the proliferation rate of CD4 SP Treg was increased in the thymus (Fig. S1A). The changes in Treg proliferation were accompanied by a continuous increase in the proliferation rate of Tcon in the peripheral lymphoid organs and in the peripheral blood that was not observed in age-matched BALB/c mice (Fig. S1B). Additionally, and in contrast to BALB/c mice, the majority of proliferating CD4+ T cells consisted of CD44+ effector/memory T cells in lupus mice (Fig. S1C). The calculated ratio between proliferating CD4+Foxp3+ Treg and CD4+Foxp3 Tcon—representing a measure of the homeostatic balance between Treg and Tcon—was therefore progressively reduced in the lymphoid organs from (NZBxNZW) F1 mice compared to that of age-matched BALB/c mice (Fig. 1C). Consistent with the increased proliferation of CD4 SP Treg, an increased Treg:Tcon ratio was observed in the thymus (Fig. 1C). Collectively, these data indicated an acquired impairment of the homeostatic balance between Treg and effector Tcon in parallel to a partial deficiency of Treg in the periphery.

Phenotypic Changes of Treg and Tcon During Disease Development.

Next, we analyzed the phenotype of CD4+Foxp3+ Treg and CD4+Foxp3 Tcon from lupus-prone (NZBxNZW) F1 mice during the course of disease. The percentage of CD25+ cells among Treg decreased with advancing disease in (NZBxNZW) F1 mice (Fig. 2A and Fig. S2A). This was accompanied by an increase in the percentage of CD25+ cells among Tcon (Fig. 2A and Fig. S2A). Compared to age-matched BALB/c mice, young (NZBxNZW) F1 mice already had higher frequencies of CD69+ and CD44+ cells among both Treg and Tcon that strongly increased with disease progression (Fig. 2 B and D and Fig. S2B and D). In contrast, the percentage of CD62L+ cells among Treg and Tcon markedly declined concomitantly with the increase in CD44+ cells (Fig. 2C and Fig. S2C). Similar phenotypic changes were present in the parental autoimmune-prone NZB strain that develops a milder form of lupus. In contrast, these phenotypic changes were not observed in the clinically healthy parental NZW strain, except for a moderate activation, or the healthy BALB/c strain (Fig. 2 and Fig. S2). Together, Treg and Tcon from lupus-prone mice acquire phenotypic changes similar to those previously described in IL-2-deficient mice (9).

Fig. 2.
Phenotypic changes of Treg and Tcon during disease progression. Average expression of CD25 (A), CD69 (B), CD62L (C), and CD44 (D) on CD4+Foxp3+- and CD4+Foxp3-gated cells from spleens of BALB/c (white bars), NZW (light gray bars), NZB (black ...

Progressive Decline in IL-2-Producing CD4+ T Cells.

To assess whether lupus-prone mice develop IL-2 deficiency, we determined the percentage of CD4+ T cells that are capable to produce IL-2 and IFN-γ during disease development in (NZBxNZW) F1 mice. In comparing young, healthy (NZBxNZW) F1 and age-matched BALB/c mice, we did not observe substantial differences in the percentage of IL-2-producing CD4+ T cells in either spleens or lymph nodes (Fig. 3 A and B). Consistent with our observation of the acquired predominance of effector/memory Tcon, CD4+ T cells acquired the ability to produce IFN-γ as the disease progressed (Fig. 3 A and B). In parallel, CD4+ T cells lost the ability to produce IL-2 (Fig. 3 A and B). Moreover, the IFN-γ-producing CD4+ T cells from (NZBxNZW) F1 mice exhibited diminished IL-2 production compared to BALB/c mice (Fig. 3 A and B). To confirm the shortage of IL-2 in lymphoid organs, we determined the spontaneous IL-2 secretion in 24-h cell cultures from spleen and lymph node cells at different disease stages. In addition, we determined IL-2 levels in the plasma during disease progression. In line with the decline of CD4+ IL-2-producing T cells, spontaneous IL-2 secretion in lymphoid organs from (NZBxNZW) F1 mice was also progressively diminished from the disease onset compared to age-matched BALB/c mice in parallel to decreasing IL-2 levels in the plasma (Fig. 3 CE).

Fig. 3.
Decline in IL-2-producing CD4+ T cells. Representative dot plots show the percentage of IL-2- and IFN-γ-producing cells among CD4+-gated spleen (A) and lymph node (B) cells from 6- to 9-month-old BALB/c and diseased (NZBxNZW) F1 mice (NZBW). Numbers ...

Treg from Lupus-Prone Mice Are Functionally Intact and Influence Disease Course.

Loss of suppressive function of Treg results in the appearance of systemic autoimmune syndromes (1, 2). We found that Treg of both young and diseased (NZBxNZW) F1 mice displayed similar suppressive functions in vitro (Fig. 4A) and similar Foxp3 mRNA expression levels compared to age-matched BALB/c mice (Fig. S3A).

Fig. 4.
Treg are functionally intact and influence disease progression. (A) In vitro suppression assays compare the suppressive capacity of CD4+CD25+ Treg from (NZBxNZW) F1 and age-matched BALB/c mice. The proliferation of CFDA-SE-labeled responder cells in the ...

To gain insights into the role of Treg in preventing disease development, we reduced the numbers of Treg in young (NZBxNZW) F1 mice with a single injection of depleting antibodies against CD25. This resulted in a transient (≈2 weeks) contraction of the CD4+Foxp3+ Treg pool in the peripheral blood (Fig. S3B), was followed by higher frequencies of CD4+CD44+ Tcon in the peripheral blood (Fig. S3B), and resulted in an acceleration of disease progression indicated by the development of nephritis ≈4–6 weeks earlier than controls (Fig. 4B). Consistent with these observations, the Treg-depleted mice also had a reduced survival rate (Fig. 4B). However, no acceleration of anti-ds-DNA-antibody titers was observed (Fig. S3B).

To test whether increasing the size of the peripheral Treg pool could influence already established disease, purified CD4+Foxp3+CD25+ Treg were adoptively transferred into mice with active disease. Because the IL-2-deficient environment in diseased animals would affect the survival and metabolic fitness of the transferred Treg, Treg were activated for 24 h in the presence of high doses of IL-2 before transfer (Fig. S3C). Adoptive transfer of Treg delayed disease progression and significantly increased the survival time (Fig. 4C). Again, there was no significant change observed in the anti-ds-DNA IgG antibody titers at any time point analyzed (Fig. S3D). Thus, CD4+Foxp3+ Treg from lupus-prone mice were functionally intact and physiologically relevant inhibitors of disease progression. Moreover, these data suggested that efficient regulation of CD4+ Tcon activation and disease progression in lupus-prone mice depended on the presence of sufficient numbers of Treg.

IL-2 Is Required to Retain Treg:Tcon Balance and to Impede Disease Progression.

To explore the role of IL-2 in the pathogenesis of murine lupus, we reduced systemic IL-2 levels with neutralizing antibodies in young and clinically healthy (NZBxNZW) F1 mice. This induced a strong and persistent reduction in the percentage of CD4+Foxp3+CD25+ Treg in the peripheral blood (Fig. S4A), in the lymph nodes (Fig. 5A), and later also in the spleen (Fig. 5A). In contrast, the percentage of CD4+Foxp3+ Treg that lacked expression of CD25 remained either unaffected 1–3 weeks after IL-2 neutralization or even considerably increased in the spleen and in the peripheral blood at later time points (Fig. S4 BD). Thus, IL-2 deficiency affected nearly exclusively the CD25+ subpopulation of Foxp3+ Treg with differing kinetics and impacts in the peripheral blood, in the lymph nodes, and in the spleen, respectively. In parallel, we observed a continuous increase of CD69+ and CD44+ cells among both the Treg and Tcon in the lymphoid organs (Fig. 5B) and of CD44+ Tcon in the peripheral blood (Fig. S4E), but we did not observe an increase in anti-ds-DNA-IgG titers (Fig. S4F). Thus, IL-2 deficiency induced similar abnormalities in Treg and Tcon that are present in untreated mice with advanced disease. Neutralization of IL-2 also resulted in a strong acceleration of nephritis (Fig. 5C) and significantly increased mortality compared to isotype-treated control mice (Fig. 5D). Together, IL-2 was necessary to maintain a pool size of Treg in the periphery that was competitive enough to prevent Tcon hyperactivity and disease progression in lupus-prone mice.

Fig. 5.
IL-2 neutralization induces Treg:Tcon imbalance and accelerates disease. (A and B) Flow cytometry of CD4+-gated T cells 21 and 42 days after injection of anti-IL-2 antibodies (anti-IL-2; filled bars) into 9- to 10-week-old, clinically healthy (NZBxNZW) ...

IL-2 Partially Restores Treg:Tcon Balance and Ameliorates Established Disease.

We next examined whether IL-2 was capable of influencing the endogenous Treg pool in (NZBxNZW) F1 mice. Treatment of onset and diseased lupus mice with recombinant IL-2 (rIL-2) strongly promoted the expansion of endogenous CD4+Foxp3+ Treg in spleens (1.5-fold; Fig. 6A), lymph nodes (1.5-fold; Fig. S5A), and peripheral blood (2-fold; Fig. S5A) compared to the PBS-treated control mice. In parallel, Treg displayed a remarkable increase in the surface expression of CD25 (Fig. 6A) and an increase in the expression of Foxp3 on a per cell basis in rIL-2-treated mice (Fig. S5B). Analysis of BrdU incorporation in vivo revealed a 2- to 3-fold increase in the proliferative activity of Treg in rIL-2-treated versus untreated mice (Fig. 6B). Although an increased proliferation of Tcon was also induced by administration of rIL-2, proliferation of Treg was favored as shown by the higher ratio between BrdU+ Treg and BrdU+ Tcon (Fig. 6B). Short-term rIL-2-treatment (3× 2 μg/24 h) of diseased lupus mice already delayed disease progression (Fig. S5C) and significantly decreased mortality (Fig. S5D); however, it did not lead to a persistent expansion of the Treg pool (Fig. S5E). In contrast, repetition of rIL-2 injections in intervals of 5 days after the initial short-term treatment for a total of four times resulted in a much more stable Treg expansion, especially of CD25+ Treg, which was still detectable >10 days after the last rIL-2 injection (Fig. S5 FH). In addition, the repetitive rIL-2 regimen efficiently impeded disease progression (Fig. 6C) and considerably prolonged the survival of diseased mice (Fig. 6D). Together, these data indicated that the acquired IL-2 deficiency could be compensated with exogenous rIL-2 and that the resulting re-establishment of the Treg:Tcon balance was associated with a beneficial outcome in murine lupus.

Fig. 6.
rIL-2 restores Treg:Tcon balance and impedes disease progression. (A) Flow cytometry of CD4+ T cells from spleens of 5-month-old (NZBxNZW) F1 mice 12 h after three injections of 2 μg of rIL-2 per mouse within 24 h (rIL-2) compared to PBS-treated ...

Discussion

Perturbations in Treg biology can elicit autoimmune syndromes and are associated with certain autoimmune diseases. Here we analyzed CD4+Foxp3+ Treg in the (NZBxNZW) F1 model of SLE to identify and characterize disturbances in Treg biology and investigate how they contributed to the pathogenesis of this systemic autoimmune disease.

Identification of Treg by expression of Foxp3 revealed a partial deficiency in the numbers of Treg in (NZBxNZW) F1 lupus mice. This deficiency affected lymph nodes and the peripheral blood, and was progressive during disease development. Along with the partial deficiency, we found a diminished homeostatic proliferation of Treg in lupus-prone mice at the onset of disease. This contrasted with a progressive increase in the proliferation of effector Tcon starting from the onset of disease, indicating a progressive homeostatic imbalance between Treg and effector Tcon. These data also suggested that the peripheral pool of Treg failed to expand adequately enough to compete with the enhanced proliferation of Tcon, a condition similar to that recently described in IL-2-deficient mice (9, 10). Phenotypic analyses revealed further similarities between IL-2-deficient and diseased (NZBxNZW) F1 lupus mice, including a progressive reduction in the expression of CD25 in Treg, the activated, memory-like phenotype of Treg, and the progressive increase in CD44+CD62L effector Tcon that predominately belong to the Th1 lineage (7, 9). Indeed, we could confirm a state of IL-2 deficiency in lupus-prone mice by the detection of a progressive deficiency of IL-2 and IL-2-producing Tcon in the lymphoid tissues. This deficiency was characterized by a decline in single IL-2-producing Tcon, which predominantly belong to the naïve Tcon repertoire (5), and a diminished IL-2 production by IFN-γ+ effector Tcon. The decline in IL-2 levels and IL-2-producing Tcon was associated with progressive Treg abnormalities and disease progression, suggesting that an acquired IL-2 deficiency contributes to disease development. This was proven by several experiments. First, neutralization of IL-2 in young, clinically healthy animals induced the homeostatic imbalance of Treg and effector Tcon, a characteristic of advanced disease, and strongly accelerated lupus. Second, treatment of diseased lupus mice with rIL-2 partially restored the balance of Treg and effector Tcon by preferentially increasing the proliferation of Treg, and considerably impeded disease progression, and the efficacy was further enhanced by a repetitive regimen.

Nonetheless, treatment with rIL-2 also resulted in an increased activation and proliferation of CD4+Foxp3 Tcon. Although this may carry the risk of increasing disease activity (22, 23), our data show that in the active phase of the disease and in an IL-2-deprived environment, IL-2 acted as a Treg adjuvant that outweighed its simultaneous fueling of the Tcon pool. Apart from that, it was recently shown in a model of type 1 diabetes that the potency of IL-2 to increase the activation of Tcon and NK T cells depended on the administered dosage (23). In line with our data, recovery from autoimmunity was also observed in the MRL/lpr lupus mouse model after treatment with IL-2-expressing recombinant vectors (24, 25). Therefore, therapeutic strategies that target the Treg-IL-2 axis as a treatment for SLE are worth exploring in more detail. Further research should aim to improve the selectivity of IL-2 treatments to favor exclusively Treg proliferation.

The pathogenic relevance of the impaired Treg-IL-2 axis in murine lupus, shown here, raises the question of the origin of the IL-2 deficiency. Both genetic and acquired alterations have been considered important for the IL-2 deficiency observed in lupus and other autoimmune diseases (2628). In our study, the deficiency of IL-2 and IL-2-producing CD4+ Tcon in the (NZBxNZW) F1 lupus model was not evident before the onset of disease and did not precede Tcon hyperactivity and Treg deficiency in general. The low prevalence of CD4+Foxp3+ Treg in young, clinically healthy lupus mice also suggests that factors other than IL-2 may be involved in regulating the size of the peripheral Treg pool. This is supported by recent studies in congenic mouse strains related to the (NZBxNZW) F1 strain that indicated that the low prevalence of CD25+ Treg was linked to a disease-related locus, the so-called Sle1a locus (29). Although the responsible genes have not been determined so far, it is known that the Sle1a locus does not encode for genes involved in IL-2 transcription or IL-2 signaling (29). Alternatively, the peripheral Treg deficiency may be caused by an impaired thymic Treg generation. Instead, we found that the numbers and proliferation rates of thymic Treg were normal in young (NZBxNZW) F1 mice and even increased during disease progression pointing to a mechanism that attempts to compensate for the Treg deficiency in the peripheral lymphoid organs. Together, we propose that IL-2 deficiency in lupus is secondary, acquired mainly because of the displacement of naïve, IL-2-producing Tcon by effector Tcon. The IL-2 deficiency arises because effector Tcon do not contribute sufficiently to overall IL-2 production. Thus, the IL-2 deficiency in lupus may be the result of an uncontrolled Tcon hyperactivity.

In contrast to the progressive deficiency observed in the peripheral blood and lymph nodes, a continuous increase of the Treg subpopulation that lacked expression of CD25 was observed in the spleen, leading to a >4-fold increase in the absolute numbers in diseased (NZBxNZW) F1 mice compared to young (NZBxNZW) F1 and aged-matched BALB/c mice. At present, it is not entirely clear why there are such differences in the distribution of Treg in this lupus model. However, the diminished or moderate in vivo proliferation rates observed in splenic Treg suggest that an accumulation of Treg in this highly inflamed tissue is more likely than an expansion in the organ itself. Therefore, the partial deficiency of Treg found in lymph nodes and in the peripheral blood may also be, in part, the result of an enhanced attraction of Treg to the spleen. Interestingly, an increase of splenic Foxp3+ Treg that lacked expression of CD25 was induced in healthy lupus mice by neutralization of IL-2, suggesting that IL-2 deficiency plays an important role in this phenomenon.

Taking all our results into consideration, we propose a simplified model that may explain the gradually acquired and self-amplifying failure of the CD4+Foxp3+ Treg pool to compete in systemic autoimmunity (Fig. S6). In this model, several initial events lead to Tcon hyperactivity. Early in the disease, at least partial compensation is provided by a qualitatively intact yet quantitatively already restricted Treg system. The origins of spontaneous Tcon hyperactivity in lupus are currently speculative but could include genetic alterations in antigen-presenting cells that lead to an increase in proinflammatory cytokine production or an altered context of self-antigen presentation to autoreactive Tcon (30). This may also promote resistance of autoreactive Tcon to Treg-mediated suppression, additionally facilitating their activation, differentiation, and consecutive expansion (31). With disease progression, the ongoing conversion of naive Tcon into effector Tcon and their displacement results in a progressive decline in IL-2 production. This acquired IL-2 deficiency further impedes the peripheral Treg pool to expand sufficiently to compete with the hyperactivity of Tcon. The resulting vicious cycle is characterized by a progressive homeostatic imbalance between Treg and effector Tcon. Once the Treg pool declines below a critical size, the remaining compensatory mechanisms collapse and the disease manifests itself.

In summary, an acquired and self-amplifying impairment of the Treg-IL-2 axis induced a progressive homeostatic imbalance of Treg and effector Tcon, and promoted the development of disease. The reversibility of this homeostatic impairment, shown here, provides rationales and approaches for a Treg- and IL-2-based immunotherapy of SLE.

Materials and Methods

See SI Materials and Methods for details.

Mice.

Female (NZBxNZW) F1, NZW, NZB, and BALB/c mice were bred and maintained under specific-pathogen-free conditions in the Deutsches Rheumaforschungszentrum (DRFZ) and were used for experiments at between 6 weeks and 12 months of age in accordance with institutional and federal guidelines. Mice were divided into groups according to age and disease activity, as determined by the degree of nephritis (young, onset, and diseased). Proteinuria was determined with Multistix 10 Visual (Bayer Diagnostics).

Flow Cytometry.

Multicolor flow cytometry was performed with a FACSCalibur cytometer (BD Biosciences). Results were processed by using CellQuest software (BD Biosciences). FACSDiva (BD Biosciences) and FACSAria (BD Biosciences) cell sorters were used for cell sorting.

In Vivo Proliferation Analysis.

Mice of different age groups were injected i.p. every 24 h for 4 days with 40 mg/kg bodyweight BrdU (BD Biosciences). BrdU incorporation was analyzed by flow cytometry (BrdU Flow kit; BD Biosciences).

IL-2 Production and ELISA.

IL-2 concentrations in cell culture supernatants from spleens and lymph nodes and in the plasma of (NZBxNZW) F1 and BALB/c mice at different ages were determined by ELISA (IL-2 ELISA kit; BD Biosciences). The concentration of anti-ds-DNA antibodies in the plasma was determined by ELISA.

Suppression Assays.

CD4+CD25+ and CD4CD25 cells were obtained from (NZBxNZW) F1 mice and age-matched BALB/c mice by MACS (Miltenyi Biotec). CD4+CD25 T cells from young syngenic mice were used as responder cells in all experiments. The proliferation of CFDA-SE-labeled responder cells in the presence of titrated numbers of CD4+CD25+ cells was determined by flow cytometry.

Adoptive Transfer of Treg.

Purified and activated CD4+CD25+Foxp3+ Treg from young (NZBxNZW) F1 mice were injected i.v. into (NZBxNZW) F1 mice at the age of between and 6.5 and 7.5 month (0.5–1.0 × 106 cells per mouse).

Treg Depletion and IL-2 Neutralization.

(NZBxNZW) F1 mice at the age of 8–15 weeks received 100 μg of depleting antibodies against CD25 (eBioscience PC61) i.v. or 1 mg of neutralizing anti-IL-2 antibodies (DRFZ, S4B6.1) by i.p. injection as previously described in other mouse strains (11); age-matched controls received either 100 μg of rat IgG1 (R&D Systems 43414.1) or 1 mg of rat IgG (Jackson ImmunoResearch), respectively.

rIL-2 Treatment.

Mice at different ages received 2 μg of rmIL-2 (R&D Systems) in PBS or PBS alone i.p. every 12 h within 24 h (three times in total; short-term treatment) 12–96 h before analysis. For survival experiments, (NZBxNZW) F1 mice with established disease either were treated with a short-term treatment of rIL-2 alone or the treatment was repeated every 5 days with 2 μg of rIL-2 for a total of four times after the initial short-term treatment (repetitive treatment).

Statistical Analysis.

GraphPad Prism 4 software was used for the analysis of survival curves (log-rank test, Kaplan–Meier curve). The Mann–Whitney U test (two-tailed) was used to detect statistical significant differences. P values of <0.05 were regarded as significant.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Hyun-Dong Chang for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 650) and grants from the University Hospital Charité Berlin.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903158107/DCSupplemental.

This article is a PNAS Direct Submission.

References

1. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4:330–336. [PubMed]
2. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol. 2005;6:345–352. [PubMed]
3. Kim JM, Rasmussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol. 2007;8:191–197. [PubMed]
4. Costantino CM, Baecher-Allan CM, Hafler DA. Human regulatory T cells and autoimmunity. Eur J Immunol. 2008;38:921–924. [PMC free article] [PubMed]
5. Malek TR. The biology of interleukin-2. Annu Rev Immunol. 2008;26:453–479. [PubMed]
6. Sadlack B, et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell. 1993;75:253–261. [PubMed]
7. Sadlack B, et al. Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells. Eur J Immunol. 1995;25:3053–3059. [PubMed]
8. Suzuki H, et al. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. Science. 1995;268:1472–1476. [PubMed]
9. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol. 2005;6:1142–1151. [PubMed]
10. D’Cruz LM, Klein L. Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nat Immunol. 2005;6:1152–1159. [PubMed]
11. Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med. 2005;201:723–735. [PMC free article] [PubMed]
12. Brandenburg S, et al. IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells. Eur J Immunol. 2008;38:1643–1653. [PubMed]
13. Malek TR, Yu A, Vincek V, Scibelli P, Kong L. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity. 2002;17:167–178. [PubMed]
14. Kotzin BL. Systemic lupus erythematosus. Cell. 1996;85:303–306. [PubMed]
15. Riemekasten G, Hahn BH. Key autoantigens in SLE. Rheumatology (Oxford) 2005;44:975–982. [PubMed]
16. Miyara M, et al. Global natural regulatory T cell depletion in active systemic lupus erythematosus. J Immunol. 2005;175:8392–8400. [PubMed]
17. 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;177:1451–1459. [PubMed]
18. Alexander T, et al. Depletion of autoreactive immunologic memory followed by autologous hematopoietic stem cell transplantation in patients with refractory SLE induces long-term remission through de novo generation of a juvenile and tolerant immune system. Blood. 2009;113:214–223. [PubMed]
19. Linker-Israeli M, et al. Defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus (SLE) J Immunol. 1983;130:2651–2655. [PubMed]
20. Dauphinée MJ, Kipper SB, Wofsy D, Talal N. Interleukin 2 deficiency is a common feature of autoimmune mice. J Immunol. 1981;127:2483–2487. [PubMed]
21. Theofilopoulos AN, Dixon FJ. Murine models of systemic lupus erythematosus. Adv Immunol. 1985;37:269–390. [PubMed]
22. Knoechel B, Lohr J, Kahn E, Bluestone JA, Abbas AK. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J Exp Med. 2005;202:1375–1386. [PMC free article] [PubMed]
23. Tang Q, et al. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity. 2008;28:687–697. [PMC free article] [PubMed]
24. Gutierrez-Ramos JC, Andreu JL, Revilla Y, Viñuela E, Martinez C. Recovery from autoimmunity of MRL/lpr mice after infection with an interleukin-2/vaccinia recombinant virus. Nature. 1990;346:271–274. [PubMed]
25. Huggins ML, Huang FP, Xu D, Lindop G, Stott DI. Modulation of autoimmune disease in the MRL-lpr/lpr mouse by IL-2 and TGF-beta1 gene therapy using attenuated Salmonella typhimurium as gene carrier. Lupus. 1999;8:29–38. [PubMed]
26. Crispin JC, Alcocer-Varela J. Interleukin-2 and systemic lupus erythematosus—fifteen years later. Lupus. 1998;7:214–222. [PubMed]
27. Yamanouchi J, et al. Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat Genet. 2007;39:329–337. [PMC free article] [PubMed]
28. Juang YT, et al. Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J Clin Invest. 2005;115:996–1005. [PMC free article] [PubMed]
29. Cuda CM, Wan S, Sobel ES, Croker BP, Morel L. Murine lupus susceptibility locus Sle1a controls regulatory T cell number and function through multiple mechanisms. J Immunol. 2007;179:7439–7447. [PubMed]
30. Baccala R, Hoebe K, Kono DH, Beutler B, Theofilopoulos AN. TLR-dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity. Nat Med. 2007;13:543–551. [PubMed]
31. 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;178:271–279. [PubMed]

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