Article Text

Extended report
Low-dose interleukin-2 selectively corrects regulatory T cell defects in patients with systemic lupus erythematosus
  1. Caroline von Spee-Mayer1,
  2. Elise Siegert1,
  3. Dimas Abdirama1,
  4. Angelika Rose1,
  5. Anika Klaus1,
  6. Tobias Alexander1,
  7. Philipp Enghard2,
  8. Birgit Sawitzki3,
  9. Falk Hiepe1,
  10. Andreas Radbruch4,
  11. Gerd-Rüdiger Burmester1,
  12. Gabriela Riemekasten1,5,
  13. Jens Y Humrich1,4
  1. 1Department of Rheumatology and Clinical Immunology, Charité—University Medicine Berlin, Berlin, Germany
  2. 2Department of Nephrology and Intensive Care Medicine, Charité—University Medicine Berlin, Berlin, Germany
  3. 3Institute of Medical Immunology, Charité—University Medicine Berlin, Berlin, Germany
  4. 4German Rheumatism Research Center (DRFZ), a Leibniz Institute, Berlin, Germany
  5. 5Department of Rheumatology, University Hospital Schleswig-Holstein—Campus Lübeck, Lübeck, Germany
  1. Correspondence to Professor Gabriela Riemekasten, Department of Rheumatology, University Hospital Schleswig-Holstein—Campus Lübeck, Ratzeburger Allee 160, Lübeck 23538, Germany; Gabriela.Riemekasten{at}uksh.de

Abstract

Objectives Defects in regulatory T cell (Treg) biology have been associated with human systemic autoimmune diseases, such as systemic lupus erythematosus (SLE). However, the origin of such Treg defects and their significance in the pathogenesis and treatment of SLE are still poorly understood.

Methods Peripheral blood mononuclear cells (PBMC) from 61 patients with SLE and 52 healthy donors and in vitro IL-2 stimulated PBMC were characterised by multicolour flow cytometry. Five patients with refractory SLE were treated daily with subcutaneous injections of 1.5 million IU of human IL-2 (aldesleukin) for five consecutive days, and PBMC were analysed by flow cytometry.

Results Patients with SLE develop a progressive homeostatic dysbalance between Treg and conventional CD4+ T cells in correlation with disease activity and in parallel display a substantial reduction of CD25 expression on Treg. These Treg defects resemble hallmarks of IL-2 deficiency and lead to a markedly reduced availability of functionally and metabolically active Treg. In vitro experiments revealed that lack of IL-2 production by CD4+ T cells accounts for the loss of CD25 expression in SLE Treg, which could be selectively reversed by stimulation with low doses of IL-2. Accordingly, treatment of patients with SLE with a low-dose IL-2 regimen selectively corrected Treg defects also in vivo and strongly expanded the Treg population.

Conclusions Treg defects in patients with SLE are associated with IL-2 deficiency, and can be corrected with low doses of IL-2. The restoration of endogenous mechanisms of immune tolerance by low-dose IL-2 therapy, thus, proposes a selective biological treatment strategy, which directly addresses the pathophysiology in SLE.

  • Systemic Lupus Erythematosus
  • T Cells
  • Cytokines
  • Treatment
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Introduction

Systemic autoimmune diseases such as systemic lupus erythematosus (SLE) are caused by a failure of endogenous mechanisms of immune tolerance. Regulatory T cells (Treg), expressing the transcription factor Foxp3, are pivotal for maintaining peripheral self-tolerance and controlling autoimmunity by suppressing the activation and expansion of autoreactive T cells and other pathogenic immune cells.1–4 The survival, growth and homeostasis of Treg fundamentally depend on the availability of the cytokine interleukin-2 (IL-2).5 ,6 Accordingly, IL-2 deficiency results in a profound disturbance of Treg homeostasis and the development of a severe systemic autoimmune disease due to uncontrolled hyperactivity of T and B cells.5–8

In SLE, the breakdown of immune tolerance predominantly towards nuclear autoantigens leads to aberrant activation of autoreactive T and B cells and the production of autoantibodies, which ultimately results in severe autoimmunity with diverse clinical manifestations,9 resembling IL-2 deficiency in many aspects. Several studies have analysed quantitative and qualitative aspects of Treg in SLE, suggesting disease-associated disturbances in Treg biology (reviewed in ref. 10). In addition, impaired IL-2 production by T cells has been associated with murine and human SLE.11–14 In previous studies, we have proven the causal involvement of an acquired IL-2 deficiency and related Treg defects in the pathogenesis of murine lupus. These Treg defects were principally characterised by a progressive homeostatic imbalance between Treg and conventional T cells (Tcon) and a concomitant loss of CD25 expression on Treg, while their suppressive function was found to be intact.15 Accordingly, we demonstrated that compensation of IL-2 deficiency by IL-2 therapy corrects associated Treg defects and ameliorates established disease in lupus-prone mice.15

To proceed to a clinical translation of IL-2 therapy for SLE, we addressed here whether lack of IL-2 is causative for Treg defects also in human SLE and whether those defects can be corrected by the supplementation of IL-2.

Methods

Procedures and reagents

All procedures and reagents used for cell separation, flow cytometry, DNA methylation analysis and in vitro experiments, including IL-2 quantification and real-time PCR analysis, IL-2 neutralisation, IL-2 stimulations, analysis of cytokine production, Treg suppression assays and statistical analyses are described in detail in the online supplementary material, Methods section.

Study subjects

Peripheral blood mononuclear cells (PBMC) were obtained from 61 patients with SLE and 52 age-matched and sex-matched healthy controls after informed consent and approval by the Institutional Review Board of the Charité—University Medicine Berlin (EA 1/342/12 and EA 1/098/07). Clinical characteristics, age, disease duration, serum levels of anti-dsDNA antibodies, disease activity according to the SLE Disease Activity Index (SLEDAI), as well as immunosuppressive treatments were retrieved from the patients’ medical records determined at the time of blood drawing, and are summarised in online supplementary table S1

Low-dose IL-2 treatment of patients with SLE

The therapeutic regimen consisted of daily subcutaneous injections of recombinant human IL-2 (aldesleukin) at a single dose of 1.5 million IU for five consecutive days. Cells from peripheral blood were analysed by flow cytometry daily before the IL-2 injections and one day after the 5th IL-2 injection. Written informed consent was obtained from the patients prior to the initiation of the off-label treatment or the clinical study with IL-2 in accordance to the Declaration of Helsinki in the revised version of 1996, and the International Conference on Harmonisation guidelines on good clinical practice. Treatments of patient 1 and 2 were announced as ‘off-label’ therapy (‘Individuelle Heilversuche’) to the Institutional Ethics Committee of the Charité—University Medicine Berlin. Patients 3–5 were treated within a phase I/IIa clinical trial, addressing the safety, tolerability, efficacy and immunological responses of a low-dose therapy with recombinant human IL-2 (aldesleukin) in the treatment of SLE (PRO-IMMUN; EudraCT-Number: 2013-001599-40; DRKS-ID: DRKS00004858). This clinical study was approved by the responsible ethics committee (EC) (Ethik-Kommission des Landes Berlin, Germany; 13/0449—EK10) and authorised by the competent governmental authority (Bundesinstitut für Arzneimittel und Medizinprodukte—BfArM, Germany; 61-3910-4039436).

Results

Active SLE is associated with an imbalanced Treg/Tcon homeostasis

A major consequence of IL-2 deficiency is a profound disruption of Treg homeostasis due to lack of growth and survival signals.5 ,6 ,15 In lupus-prone mice, we previously identified an imbalanced homeostatic proliferation between Treg and Tcon, which was progressive during disease development and which could be linked to an acquired IL-2 deficiency.15 Thus, we performed ex vivo analyses of the proliferation marker Ki67 in CD3+CD4+Foxp3+CD127lo Treg and CD3+CD4+Foxp3- Tcon, which revealed no significant differences in the proliferation rates of Treg between patients with SLE and healthy donors. However, in analogy to murine lupus, significantly higher percentages of proliferating Tcon in patients with SLE compared with healthy controls were observed (figure 1A). The frequency of proliferating Tcon correlated with the SLEDAI and serum levels of anti-dsDNA antibodies in patients with SLE (see online supplementary figure S1A). Calculation of the ratio between proliferating Treg and proliferating Tcon, as a measure of the homeostatic Treg/Tcon balance, revealed a substantially lower Treg:Tcon proliferation ratio in patients with SLE compared with healthy donors (median 3.3 vs 8.4; figure 1B), which inversely correlated with the SLEDAI and anti-dsDNA antibody levels (figure 1C). Moreover, higher percentages of Treg from patients with SLE were positive for active Caspase-3, a marker for apoptotic cells (figure 1D), and expressed low levels of the antiapoptotic protein Bcl-2 (see online supplementary figure S1C). Although Tcon from patients with SLE also contained higher proportions of active Caspase-3+ and Bcl-2lo cells (see online supplementary figure S1B,C), the higher rate of apoptosis among Tcon was clearly associated with their increased proliferation, suggesting that Tcon undergo activation-induced cell death (figure 1D, F). By contrast, no or only marginal such correlations were observed in Treg from patients with SLE (see online supplementary figure S1D, F).

Figure 1

Imbalanced Treg/Tcon homeostasis in correlation with disease activity in patients with SLE. (A) Representative overlay dot plots show the expression of Ki67 among CD3+CD4+Foxp3- Tcon (grey) and among CD3+CD4+Foxp3+CD127lo Treg (blue) from HC and patients with SLE determined ex vivo by flow cytometry. Scatter plots show the percentages of Ki67+ cells among CD3+CD4+Foxp3+CD127lo Treg (left) and among CD3+CD4+Foxp3- Tcon (right). (B) Calculated ratio between the frequency of Ki67+ Treg and of Ki67+ Tcon (Treg:Tcon proliferation ratio) from HC and patients with SLE. (C) Inverse correlation of the Treg:Tcon proliferation ratio from patients with SLE with the SLEDAI (left) and with serum levels of anti-dsDNA antibodies (right). (D) Frequencies of active Caspase-3+ cells among CD3+CD4+Foxp3+CD127lo Treg from HC and patients with SLE. Numbers in dot plots indicate the percentage of the designated population among the respective parent population. Horizontal lines in scatter plots indicate the median. Mann–Whitney test was used to compare HC (n=52) and SLE samples (n=61), correlations were assessed by Spearman's rank correlation coefficients (r) (**p<0.01, ***p<0.001). HC, healthy control; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; Tcon, conventional T cells; Treg, regulatory T cells.

Together, these data point to a relevant contribution of an imbalanced Treg/Tcon homeostasis to disease activity and suggest a deficiency of an important growth and survival factor for Treg, such as IL-2.

Treg from patients with SLE develop an IL-2 deprived phenotype in correlation with disease activity

One phenotypic hallmark of IL-2 deficiency is the loss of surface expression of CD25 on Treg.6 ,15 As previously shown by others,16 ,17 percentages of total Foxp3+CD127lo Treg among CD3+CD4+ T cells were higher in patients with SLE (figure 2A), and correlated with the SLEDAI and with serum levels of anti-dsDNA antibodies (see online supplementary figure S2A), while absolute Treg numbers and Foxp3 expression levels in Treg were not different between patients with SLE and healthy controls (see online supplementary figure S2B,C). By contrast, analysis of surface expression of CD25 on Treg from patients with SLE revealed significantly lower frequencies of Treg expressing CD25 at high and intermediate levels (CD25hi and CD25int) and an increase of CD25 negative (CD25neg) cells among Treg compared with healthy controls (figure 2B). Furthermore, absolute numbers of CD25hi Treg from patients with SLE were also significantly lower (see online supplementary figure S2D). The frequency of total CD25+ cells (CD25hi+int) among Treg correlated inversely with the SLEDAI and serum levels of anti-dsDNA antibodies in patients with SLE (figure 2C). In addition, the Treg:Tcon proliferation ratio in patients with SLE correlated with the frequency of CD25+ Treg (figure 2D), providing a linkage between the imbalanced Treg/Tcon homeostasis and the loss of CD25 expression in Treg.

Figure 2

Treg from patients with SLE acquire an IL-2-deprived phenotype. (A) Representative dot plots and scatter plot show the percentage of Foxp3+CD127lo Treg among CD3+CD4+ T cells from HC (circles) and patients with SLE (squares) analysed by flow cytometry. (B) Representative dot plots and scatter plot show the frequencies of CD25hi, CD25int and CD25neg cell subsets among CD3+CD4+Foxp3+CD127lo Treg from HC and patients with SLE. (C) Inverse correlation of the SLEDAI (left) and of serum levels of anti-dsDNA antibodies (right) with the frequency of CD25+ cells among CD3+CD4+Foxp3+CD127lo Treg. (D) Correlation of the Treg:Tcon proliferation ratio with the frequency of CD25+ cells among CD3+CD4+Foxp3+CD127lo Treg. Numbers in dot plots indicate the percentage of the designated population among the parent population. Horizontal lines in scatter plots indicate the median. Mann–Whitney test was used to compare HC (n=52) and patients with SLE (n=61), correlations were assessed by Spearman's rank correlation coefficients (r) (**p<0.01, ***p<0.001). HC, healthy control; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; Tcon, conventional T cells; Treg, regulatory T cells.

Reduced availability of functionally and metabolically active Treg in SLE

Analysis of the in vitro suppressive capacity of Treg revealed a comparable ability of CD4+CD127loCD25+ Treg to suppress Tcon proliferation between healthy donors and patients with SLE (see online supplementary figure S3A) similar to findings from other investigators in the past.10 ,16 ,18 However, these assays did not provide information about the suppressive capacity of the entire Foxp3+CD127lo Treg population, including CD25neg Treg, since it is not possible to isolate Foxp3+CD127loCD25neg cells alive for functional assays. Thus, to study whether the loss of CD25 expression in Treg from patients with SLE has an impact on Treg composition, functionality or activity, we divided Foxp3+CD127lo Treg according to their CD25 expression levels into CD25hi, CD25int and CD25neg subsets, and analysed their phenotype in more detail. All three Treg subsets comprised high frequencies of cells with a CCR7+CD45RO+ central memory phenotype. By contrast, Treg with a CCR7-CD45RO+ effector memory phenotype were mostly enriched in the CD25hi subset, while CCR7+CD45RO- naïve Treg were only found among CD25int and CD25neg Treg (figure 3A–C). In comparison with the CD25int and CD25neg Treg subsets, CD25hi Treg contained by far the highest frequencies of cells positive for CD137, which is expressed by recently antigen-activated T cells (figure 3D and see online supplementary figure S3B).19 ,20 In addition, the proportion of proliferating (Ki67+) cells was also highest in the CD25hi Treg subset, followed by the CD25neg and the CD25int Treg subset (figure 3E and see online supplementary figure S3B). Analysis of the expression of CD39, which conveys Treg-mediated suppression through extracellular ATP hydrolysis, and therefore, serves as a surrogate marker for their suppressive capacity,21 ,22 showed that most CD25hi Treg expressed CD39, while only half of the CD25int and CD25neg Treg subset were CD39+ (figure 3F and see online supplementary figure S3B). Similar distributions were also observed in Treg subsets of healthy controls (figure 3A–F and see online supplementary figure S3B). Expression of the transcription factor Helios has been associated with thymus-derived Treg and a bona fide Treg phenotype in SLE.17 ,23 ,24 In healthy controls, highest proportions of Helios+ cells were found in the CD25hi and CD25int Treg subset, while only 38% of the CD25neg Treg population expressed Helios (figure 3G and see online supplementary figure S3B,C). By contrast, in patients with SLE high proportions of Helios+ cells were observed in all Treg subsets, including the CD25neg Treg subset (figure 3G and see online supplementary figure S3B, C). In addition, we found that the proportion of cytokine-producing cells within the CD25neg Treg subset, representing contaminating Tcon, was also lower in patients with SLE compared with healthy controls, while the other Treg subsets did not contain relevant amounts of cytokine-producing cells (figure 3H, I and see online supplementary figure S3B). This suggests that the majority of Foxp3+CD127loCD25neg cells from patients with SLE consists of genuine Treg, rather than being recently activated Tcon, which can transiently express Foxp3. This was also confirmed by comparing the methylation status of the Treg-specific demethylated region (TSDR) of CD25neg and CD25+ Treg from patients with SLE (see online supplementary figure S4). In conclusion, the enrichment of CCR7-CD45RO+, CD137+, Ki67+ and CD39+ cells in the CD25hi Treg subset indicates that antigen-experienced and metabolically active Treg with a presumably high suppressive capacity are associated with high expression levels of CD25. Accordingly, loss of the CD25hi Treg subset in patients with active SLE may lead to a reduced availability of Treg that are capable of efficiently counteracting autoimmunity.

Figure 3

Enrichment of metabolically and functionally active Treg in the CD25hi Treg subset. (A–G) CD3+CD4+Foxp3+CD127lo Treg from patients with SLE (n=61) and HC (n=52) were analysed ex vivo by flow cytometry and gated according to the expression levels of CD25. Frequencies of CCR7+CD45RO- cells (A), CCR7+CD45RO+ cells (B), CCR7-CD45RO+ cells(C), CD137+ cells (D), Ki67+ cells(E), CD39+ cells (F) and Helios+ cells (G) among the CD25hi, CD25int and CD25neg Treg subsets were compared among each other and between SLE and HC samples. (H, I) Frequencies of interferon (IFN)-γ+ cells (H) and IL-2+ cells (I) among the CD25hi, CD25int and CD25neg Treg subsets were determined in sorted and polyclonally stimulated CD4+ T cells from HC (n=8) and patients with SLE (n=5). Mann–Whitney test was used to compare HC and SLE samples. Wilcoxon signed-rank test was used to analyse the differences between Treg subsets within each group. Bars represent median with IQR (*p<0.05, **p<0.01, ***p<0.001). HC, healthy control; SLE, systemic lupus erythematosus; Treg, regulatory T cells.

Treg defects in patients with SLE can be attributed to lack of IL-2

Defective IL-2 production by T cells from patients with SLE has already been reported previously,11 ,25 but has been challenged by studies showing unchanged or even increased levels of IL-2-producing CD4+ T cells after polyclonal stimulation (ref. 17, and see online supplementary figure S3D).

Therefore, we now aimed to analyse whether IL-2 deficiency is detectable in patients with SLE under physiological conditions. At first, we determined protein levels of IL-2 in sera, which, however, were below detection limits in 85% of both patients with SLE and healthy donors (data not shown). Thus, we next explored spontaneous IL-2 expression in cell cultures without adding exogenous stimuli to reflect physiological conditions. Analysis of supernatants of unstimulated PBMCs revealed significantly lower IL-2 protein levels in samples of patients with SLE compared with healthy controls after 44 h in culture (figure 4A, left). To rule out a potential bias due to low CD4+ T cell numbers commonly present in SLE samples as a result of lymphopenia, we next analysed IL-2 mRNA expression in isolated CD4+ T cells after a 24 h culture period, and found that this was significantly lower in patients with SLE compared with healthy controls (figure 4A, right). No association between IL-2 levels and the SLEDAI was observed for both mRNA and protein levels (see online supplementary figure S5). These data, however, indicate an impairment of CD4+ T cells from patients with SLE to produce normal amounts of IL-2. To investigate whether the reduced CD25 expression in Treg of patients with SLE could be attributed to the deficient spontaneous IL-2 production, surface expression of CD25 on Treg was determined in unstimulated PBMCs from patients with SLE and healthy controls after a 48 h culture period. We found that CD25 was spontaneously upregulated in Treg of healthy donors (figure 4B, left), whereas no upregulation of CD25 was observed in Treg of patients with SLE (figure 4B, right). Addition of neutralising anti-IL-2-antibodies completely inhibited the observed upregulation of CD25 expression in healthy control samples, but had no effect on Treg of SLE samples (figure 4B). These in vitro observations indicate that lack of CD25 upregulation in Treg from patients with SLE can be attributed to the impaired spontaneous IL-2 production by CD4+ T cells. Accordingly, these data suggest that a diminished in vivo availability of IL-2 accounts for the loss of the CD25hi Treg subset in patients with SLE.

Figure 4

IL-2 deficiency in patients with SLE and its effects on CD25 expression in Treg. (A) IL-2 protein levels in supernatants of unstimulated PBMCs cultured for 44 h (left) and IL-2 mRNA expression relative to the housekeeper EEF1A1 in magnetic activated cell sorting (MACS)-sorted and unstimulated CD4+ T cells after 24 h cell culture (right) from HC and patients with SLE. (B) Frequencies of CD25hi cells among Foxp3+CD127lo Treg from HC (n=11) and patients with SLE (n=7) were analysed by flow cytometry either ex vivo (white bars), or within PBMCs cultured for 48 h either alone (black bars), with the addition of neutralising anti-IL-2 antibodies (dark grey bars) or with the addition of an irrelevant control antibody (ctrl Ig, light grey bar) (mean and SD). Mann–Whitney test was used to compare HC and SLE samples. Wilcoxon signed-rank test was used to compare different culture conditions (*p<0.05, **p<0.01, ***p<0.001). HC, healthy control; SLE, systemic lupus erythematosus; Treg, regulatory T cells.

Low-dose IL-2 stimulation selectively reverses Treg defects and enhances pro-survival signals in Treg from patients with SLE

Next, we evaluated whether the observed Treg defects in patients with SLE could be reversed in vitro by stimulation with IL-2. Complete PBMCs from patients with SLE and healthy controls were repetitively stimulated every 24 h with various concentrations of recombinant human IL-2 (aldesleukin) on five consecutive days, and were analysed 24 h after the first (24 h) and the fifth (5 days) IL-2 stimulation. In order to get close to a clinical application of Treg reconstitution by low doses of IL-2, concentrations of 1.0, 2.5, 4.0 and 10.0 ng/mL were chosen according to estimated peak serum levels that are reached when subcutaneously injecting either 1.5, 3.0, 4.5 or 12.5 million IU of IL-2, respectively.26 In samples from patients with SLE, all applied IL-2 doses significantly augmented expression levels of CD25 in Treg within 24 h compared with untreated samples (figure 5A). As a consequence, significantly higher frequencies of CD25hi Treg were observed after IL-2 stimulation (figure 5B, left), and in parallel, frequencies of both CD25int and CD25neg Treg declined (figure 5B). These effects were less pronounced after repetitive stimulation for 5 days, but interestingly, the most sustained increase of CD25 expression in Treg and consecutively of CD25hi cells among Treg was induced with the lowest doses of IL-2. In contrast to Treg, IL-2 stimulation had no global effect on the expression levels of CD25 in Tcon from patients with SLE (figure 5A). The frequency of CD25hi cells among Tcon, however, increased slightly 24 h after IL-2 stimulation, but this effect was most pronounced following stimulation with the highest doses (figure 5C). In healthy control samples, IL-2 stimulation induced comparable effects in Treg and Tcon as in SLE samples (see online supplementary figure S6A, B), which, in contrast to previous studies,11 indicates an unimpaired responsiveness of SLE T cells to IL-2. No relevant increase in the proliferation of Treg or Tcon and only a moderate increase in the frequencies of total Foxp3+CD127lo Treg was induced by in vitro IL-2 stimulation (see online supplementary figure S6C–E) suggesting that the augmented frequencies of CD25hi Treg are attributable to a recruitment from the CD25neg and CD25int Treg subsets rather than to an expansion of the CD25hi Treg subset itself. This proposes that both CD25neg and CD25int Treg are capable of regaining high expression levels of CD25, and thus, can be redistributed into the CD25hi subset by IL-2 stimulation despite the previous low expression levels of CD25. Stimulation with IL-2 also strongly elevated Foxp3 expression levels in Treg (see online supplementary figure S6E), and induced a significant increase in the expression of the antiapoptotic protein Bcl-2 in Treg from patients with SLE, which was evident already with the lowest dose, and did not further increase by the dose escalation (figure 5D). In contrast, the effect of IL-2 stimulation on Bcl-2 expression in Tcon from patients with SLE was dose-dependent and generally less pronounced (figure 5D). Similar effects were observed in healthy control samples, with the exception that the increase in Bcl-2 expression in Treg was rather dose-dependent after 24 h stimulation and less prominent after 5 days of stimulation compared with patients with SLE (see online supplementary figure S6F). Repetitive IL-2 stimulation for 5 days also significantly increased the proliferation of CD3-CD56+ natural killer (NK) cells and resulted in a concomitant dose-dependent increase of CD56bright cells among NK cells (see online supplementary figure S7A, B). In addition, a slightly increased Bcl-2 expression in NK cells (see online supplementary figure S7A) and in CD3+CD56+ natural killer T (NKT) cells was observed (see online supplementary figure S7E). However, no considerable effects on the proliferation and expression levels of Bcl-2 or CD25 were observed in CD3-CD19+ B cells or CD3+CD8+ T cells (see online supplementary figure S7C, D), which can also respond to IL-2.27 ,28 Together, in vitro low-dose IL-2 stimulation selectively restored CD25 expression and enhanced pro-survival signals in Treg from patients with SLE, while only marginally affecting other immune cells.

Figure 5

Selective increase of CD25 and Bcl-2 expression in Treg on in vitro low-dose IL-2 stimulation. PBMCs from patients with SLE (n=10) were repetitively stimulated every 24 h either with none, 1.0, 2.5, 4.0 or 10.0 ng/mL of human IL-2 (aldesleukin) on five consecutive days and analysed by flow cytometry 24 h after the first (24 h) or the last (5 days) in vitro stimulation. (A) Representative histograms show the expression levels of CD25 in CD3+CD4+Foxp3+CD127lo Treg and in CD3+CD4+Foxp3- Tcon after 24 h stimulation (grey line) and after 5-day stimulation (black line) with 1 ng/mL of IL-2 compared with unstimulated samples (filled). Bar diagrams show the fold-change of the MFI of CD25 in CD3+CD4+Foxp3+CD127lo Treg (left) and in CD3+CD4+Foxp3- Tcon (right) after stimulation with different doses of IL-2 compared with untreated samples. (B and C) Bar diagrams show the percentages of CD25hi, CD25int and CD25neg cells among CD3+CD4+Foxp3+CD127lo Treg (B) and of CD25hi cells among CD3+CD4+Foxp3- Tcon (C) after stimulation with different doses of IL-2 compared with unstimulated samples. (D) Representative histograms show Bcl-2 expression in CD3+CD4+Foxp3+CD127lo Treg and in CD3+CD4+Foxp3- Tcon after 24 h (grey line) and after 5-day stimulation (black line) with 1 ng/ml of IL-2 compared with unstimulated samples (filled). Bar diagrams show the fold-change of the MFI of Bcl-2 in CD3+CD4+Foxp3+CD127lo Treg (left) and in CD3+CD4+Foxp3- Tcon (right) after stimulation with different doses of IL-2 compared with untreated samples. Bars indicate median with IQR. Wilcoxon signed-rank test was used to analyse the changes induced by the different IL-2 concentrations compared with untreated samples (asterisks above error bars) or between different IL-2 concentrations (asterisks above horizontal lines) at the respective time points. (*=p<0.05, **=p<0.01, ***=p<0.001). MFI, median fluorescence intensity; SLE, systemic lupus erythematosus; Tcon, conventional T cells; Treg, regulatory T cells.

Low-dose IL-2 therapy selectively corrects Treg defects and expands the Treg population in patients with SLE

The identification of causality between Treg defects and IL-2 deficiency in patients with SLE and the reversibility of Treg defects by in vitro stimulation with low doses of IL-2, together with our previous animal studies,15 provided a convincing rationale for the clinical translation of a low-dose IL-2 therapy in SLE. Thus, we treated five patients with refractory SLE (see online supplementary table S2) with a low-dose IL-2 regimen consisting of daily subcutaneous injections of 1.5 million IU of human IL-2 (aldesleukin) on five consecutive days in analogy to our in vitro experiments, and analysed day-to-day changes in different lymphocyte subsets. During the 5-day treatment cycle, the total number and percentage of Foxp3+CD127lo Treg among CD3+CD4+ T cells gradually increased in all patients (figure 6A). This was accompanied by a twofold to threefold increase in the expression levels of CD25 in Treg and a dramatic expansion of the CD25hi Treg subset among Foxp3+CD127lo Treg (figure 6B). No increase in CD25 expression levels was observed in Tcon, but similar to the in vitro stimulations, a slight increase in the frequency of CD25hi Tcon occurred (figure 6B). Low-dose IL-2 therapy also strongly increased the proliferation of Treg in all five patients (figure 6C). In general, we observed only a slight concomitant augmentation of Tcon proliferation, such that the Treg:Tcon proliferation ratio either increased (patient 1, 4 and 5) or was unaffected (patient 3) (figure 6C). In patient 2, however, whose Treg actually showed the highest expression levels of CD25 in response to IL-2, Tcon proliferation at days 4–6 outweighed the proliferation of Treg, resulting in a reduction of the Treg:Tcon proliferation ratio (figure 6C). In parallel, the frequency of CD39+ cells among Treg increased in all patients, and the proportion of Helios+ Treg remained stable (figure 6D), suggesting the expansion of thymus-derived Treg with a high suppressive capacity by low-dose IL-2 therapy. Analysis of other lymphocyte subsets also revealed increased proliferation rates in CD8+ T cells, NKT cells and NK cells during the 5-day treatment cycle in all patients, which, however, did not result in increased numbers or frequencies at day 6 (see online supplementary figure S8B–D). Within NK cells, proliferation was most prominently induced in the CD56bright subset leading to an increase in their frequency among NK cells during the 5-day treatment cycle in all patients (see online supplementary figure S8E). Together, these studies in human beings provided the first proof-of-concept that low-dose IL-2 therapy is capable to efficiently and selectively target the Treg population in patients with SLE.

Figure 6

Selective Treg reconstitution in patients with SLE by low-dose IL-2 therapy. PBMCs of five patients with SLE were analysed daily by flow cytometry before each IL-2 injection (days 1–5) and 1 day after the last IL-2 injection (day 6) throughout the 5-day treatment cycle with low-dose IL-2, consisting of daily subcutaneous injections of 1.5 million IU of recombinant human IL-2 (aldesleukin). Representative dot plots are shown from patient 1 before the 1st IL-2 injection (day 1) and 20 h after the last injection (day 6), with the numbers indicating the percentage of the designated population among the respective parent population. Diagrams show the individual courses of the indicated parameters from the respective patients during the treatment period. (A) Dot plots show percentages of Foxp3+CD127lo Treg among CD3+CD4+ T cells. Diagrams show the percentages of Foxp3+CD127lo Treg among CD3+CD4+ T cells and the absolute cell numbers of CD3+CD4+Foxp3- Tcon and of CD3+CD4+Foxp3+CD127lo Treg per microlitre whole blood. (B) Overlay dot plots of CD3+CD4+Foxp3- Tcon (grey) and of CD3+CD4+Foxp3+CD127lo Treg (blue) show the percentages of CD25hi, CD25int and CD25neg cells among the respective parent population. Diagrams show the percentages of CD25hi cells among CD3+CD4+Foxp3+CD127lo Treg and the MFI of CD25 in CD3+CD4+Foxp3+CD127lo Treg and in CD3+CD4+Foxp3- Tcon. (C) Overlay dot plots of CD3+CD4+Foxp3- Tcon (grey) and of CD3+CD4+Foxp3+CD127lo Treg (blue), and diagrams show the percentages of Ki67+ cells among CD3+CD4+Foxp3+CD127lo Treg and among CD3+CD4+Foxp3- Tcon, and the calculated ratio between Ki67+ Treg and Ki67+ Tcon (Treg:Tcon proliferation ratio). (D) Dot plots and diagrams show the percentages of CD39+ and of Helios+ cells among CD3+CD4+Foxp3+CD127lo Treg. MFI, median fluorescence intensity; SLE, systemic lupus erythematosus; Tcon, conventional T cells; Treg, regulatory T cells.

Discussion

In patients with SLE, we identified a homeostatic disturbance of Treg in association with disease activity, which was characterised by an imbalanced Treg/Tcon proliferation and an increased apoptosis of Treg. In parallel, we found that Treg from patients with SLE showed a profound and disease-activity-related decrease in the expression of CD25, resulting in a substantial reduction of the CD25hi Treg subset by more than one-half, which is consistent with the findings of other investigators.16 ,18 ,29 ,30 These Treg defects are similar to those observed in lupus-prone and IL-2−/− mice, and thus represent typical features of IL-2 deficiency.6 ,15 In line with this, we show here by in vitro experiments that an insufficient availability of spontaneously produced IL-2 accounts for the low CD25 expression in Treg from patients with SLE, providing a causal relationship between IL-2 deficiency and Treg defects in SLE. As shown here by phenotypic comparison of the CD25hi, CD25int and CD25neg Treg subsets, the significance of the loss of the CD25hi Treg subset due to IL-2 deficiency is that it leads to a deficiency of antigen-experienced and metabolically active Treg presumably with a high suppressive capacity, thus creating a vulnerable condition in the tolerance network.

Earlier studies suggested that Foxp3+CD25neg T cells in patients with SLE represent a subset of Foxp3-expressing Tcon.16 ,31 Yet, the high percentage of Helios-expressing cells among the Foxp3+CD127loCD25neg subset, which we exclusively found in patients with SLE, together with their relatively high TSDR demethylation and the low proportions of cytokine-producing cells among them, rather implies that the majority of these cells originates from genuine Treg. Accordingly, we conclude that the high prevalence of CD25neg Treg in patients with SLE is a result of Treg exhaustion due to IL-2 deprivation, similar to previous observations in murine lupus.15 Corresponding to this, our in vitro experiments showed that the SLE-associated abnormal Treg phenotype was reversible, as IL-2, especially when applied in low doses, selectively increased expression levels of CD25 in the entire Treg population, presumably also including the CD25neg subset.

Together, these data provided a scientific rationale and a safe basis for the clinical translation of low-dose IL-2 therapy in patients with SLE. Indeed, we show here that treatment of patients with SLE with low doses of IL-2 selectively restored CD25 expression in Treg also in vivo and induced a remarkable expansion of the Treg population, especially of the CD25hi Treg subset. The parallel increase in CD39+ Treg and the preservation of high expression levels of Helios suggest that the IL-2 expanded Treg population represents thymus-derived Treg with a high suppressive capacity.21 ,24

As expected, also Tcon, NKT cells, NK cells and CD8+ T cells responded to the IL-2 treatment as they variably express IL-2-receptor chains.26 ,27 ,32 The fact that their increased proliferation did not result in persistently higher frequencies or cell counts, however, suggests that in vivo Treg also are the preferential responders to low-dose IL-2 in patients with SLE. Apart from Treg, CD56bright NK cells showed the strongest response to in vitro and in vivo stimulations with IL-2. Interestingly, CD56bright NK cells are associated with immunoregulatory properties rather than cytotoxicity.33 Similar cellular responses were also observed during low-dose IL-2 therapy in patients with other diseases34–37 pointing to a common response pattern.

First evidence that Treg reconstitution by IL-2 is clinically effective in SLE comes from a recent report showing a rapid and robust induction of clinical remission in parallel to a remarkable expansion of the Foxp3+CD127loCD25hi Treg population in one patient with SLE who received four treatment cycles of low-dose IL-2.38 In this regard, our recently initiated clinical trial addressing the safety, tolerability, efficacy and immunological responses of a low-dose IL-2 therapy in patients with SLE will provide more profound information on the clinical efficacy and the immunological mechanisms of this novel treatment. Besides this, the identification of a relevant disturbance of the Treg-IL-2 axis in human SLE as a key feature of systemic autoimmunity and its reversibility could offer the basis for a targeted therapy also in other immune-mediated diseases in which such acquired Treg defects have been observed.10

In summary, we found that Treg are vitally affected by IL-2 deficiency in SLE. These Treg defects contribute to disease pathogenesis, and can be selectively reversed by low doses of IL-2, providing a bench-to-bedside approach to a low-dose IL-2 therapy in SLE and confirming the rationales of this selective biological treatment strategy.

Acknowledgments

We thank Philippe Saikali for critical reading of the manuscript, and Toralf Kaiser and Jenny Kirsch from the Flow Cytometry Core Facility of the DRFZ for their technical assistance.

References

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Supplementary materials

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Footnotes

  • Handling editor Tore K Kvien

  • GR and JYH contributed equally.

  • Contributors CvS-M, ES, DA, AR, AK and JYH performed the experiments and acquired the data; CvS-M, BS and JYH analysed and interpreted the data; CvS-M, GR and JYH designed the study; TA, PE, FH, ARb and G-RB assisted in the study design; CvS-M, GR and JYH wrote the manuscript and compiled the figures; TA, PE, FH, ARb, and G-RB participated in the preparation of the manuscript; GR and JYH supervised the study and contributed equally to this work.

  • Funding This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) within the Sonderforschungsbereich 650 (SFB 650) and by grants from the Charité—University Medicine Berlin, Germany. CvS-M was supported by the Ernst Schering Foundation, Berlin, Germany.

  • Competing interests None declared.

  • Ethics approval IRB Charité, BfArM, EC Berlin.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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