Introduction
Regulatory T cells (Treg) constitute on average 1 to 2% of human peripheral blood mononuclear cells (PBMC) and are characterized by their capacity to actively suppress T cell proliferation
in vitro[
1]. Treg play an important role in T cell homeostasis and are critical regulators of immune tolerance. Quantitative and/or qualitative deficiencies of Treg have been suggested to contribute to the development of autoimmune diseases [
2‐
8].
Treg are best characterized by high expression levels of the IL-2 receptor α-chain (CD25) [
9,
10]. In addition the forkhead family transcription factor (Foxp3) has been described as a highly specific intracellular marker molecule for Treg [
11‐
14]. Helios, a member of the Ikaros transcription factor family, has recently been described as a specific marker for thymic-derived Foxp3+ regulatory T cells [
15,
16]. In addition it has been shown that proportions but not absolute numbers of Foxp3
+Helios
+ T cell are increased in systemic lupus erythematosus (SLE) patients and that Foxp3
+Helios
- T cells are capable of effector cytokine production [
17].
We have recently identified a novel subset of CD4
+Foxp3
+ Treg that does not express CD25 surface molecules (CD4
+CD25
-Foxp3
+) [
18]. CD4
+CD25
-Foxp3
+ share phenotypic characteristics with conventional CD4
+CD25
+Foxp3
+ Treg and convey lower, but still considerable suppression of T cell proliferation, though not of IFN-γ production,
in vitro. Increased proportions of CD4
+CD25
-Foxp3
+ Treg are observed in particular in SLE patients, a finding that has widely been confirmed [
19‐
23]. However, the origin, the precise functional role and the potential pathogenetic involvement of CD4
+CD25
-Foxp3
+ Treg are still enigmatic.
In this study we therefore evaluated if the presence of CD4+CD25-Foxp3+ cells is associated with a particular phenotype of organ manifestations in SLE patients. Our data reveal that CD4+CD25-Foxp3+ that share phenotypic characteristics with regulatory T cells are increased in patients with lupus nephritis.
Materials and methods
Patients and controls
SLE patients (n = 61; mean age 45 ± 16.8 years) who fulfilled at least four of the revised SLE criteria of the American College of Rheumatology [
24] were randomly selected from our outpatient clinic. Healthy volunteers served as a healthy control (HC) population (n = 36; mean age 42 ± 15.7 years). The disease activity of SLE patients was assessed using the SLE disease activity index (SLEDAI) [
25], the European Consensus Lupus Activity Measurement (ECLAM) score [
26] and the SLE index score (SIS) [
27]. A detailed patient characteristic is shown in Table
1. Patients were divided into two groups according to their disease activity as determined by the SLEDAI score. A SLEDAI score ≥6 was defined as high disease activity and a SLEDAI score of <6 as low disease activity. Depending on their clinical status we divided the patients into groups with active and no active organ involvement at the time of the patient’s blood draw. Active organ involvement was defined by clinical and laboratory parameters. The following items were considered to be indicative for active organ involvement: active skin involvement defined as lupus rash, discoid lupus or photosensitivity; active joint involvement defined as arthritis with synovial swelling; active hematologic involvement defined as thrombocytopenia, lymphocytopenia, or leukocytopenia or (Coombs test positive) hemolytic anemia; active renal involvement defined as nephritis with proteinuria >0.5 g protein/24 h and/or active nephritic sediment. In all patients with renal involvement a renal biopsy had been performed and glomerulonephritis was classified according to the World Health Organization (WHO) classification [
28]. All patients with renal manifestation were classified as having glomerulonephritis WHO III or IV.
Table 1
Demographic and clinical characteristics of systemic lupus erythematosus patients
Age, years, mean ± SD | 40.4 ± 14.5 |
Gender, female/male, n (%) | 56 (91.8%)/5 (8.2%) |
Disease duration years, mean ± SD | 5.4 ± 4.9 |
Anti-ds DNA Ab-positive, n (%) | 23 (62.3%) |
C3, mean ± SD | 90.5 ± 26.6 |
C4, mean ± SD | 14.3 ± 7.1 |
CH50, mean ± SD | 110.6 ± 31.9 |
SLEDAI, mean ± SD | 4.2 ± 4.2 |
ECLAM, mean ± SD | 1.9 ± 1.9 |
SIS, mean ± SD | 3.6 ± 2.9 |
Current prednisone dose, mg/day, mean ± SD | 10.4 ± 15.3 |
Concurrent immunosuppressive therapy, n (%) | |
Hydroxychloroquine | 22 (36.1%) |
Mycophenolate mofetil | 9 (14.8%) |
Cyclophosphamide | 6 (9.8%) |
Azathioprine | 8 (13.1%) |
Methotrexate | 5 (8.2%) |
Organ involvement, n (%) | |
Skin/active | 37 (60.7.1%)/8 (13.1%) |
Hematologic/active | 23 (37.7%)/10 (16.4%) |
Arthritis/active | 22 (36.1%)/3 (4.9%) |
Serositis/active | 8 (13.1%)/2 (3.3%) |
Renal/active | 25 (40.9%)/13 (21.3%) |
Ethical approval for this study was granted by the local ethics committee of the Medical University of Vienna and internal review board of the Medical University of Vienna, Austria. Patients gave written informed consent to participate in the study and agreed that the findings of the study will be published in a scientific journal.
Antibodies
The following mAb/conjugates were used in this study: R-phycoerythrin (PE), phycoerythrin-cyanin5 (PE-Cy5) and allophycocyanin (APC), PE-Cy7-conjugated mAb against CD4 (SK3) and CD25 (2A3) were purchased from Becton Dickinson (San Jose, CA, USA); mAb against Foxp3 (236A/E7) was obtained from eBiosciences (San Diego, CA, USA); mAb against CD14 (RMO52) was obtained from Beckman Coulter (Fullerton, CA, USA), mAb against Helios (22 F6) was obtained from Biolegend (San Diego, CA, USA).
Phenotypic analyses
PBMC were isolated from heparinized blood by layering over LSM 1077 Lymphocyte Separation Medium (PAA laboratories, Pasching, Austria) and density gradient centrifugation at 400 × g. PBMC were resuspended in PBS/3% human Ig (Baxter International Inc., Vienna Austria) in order to block Fc receptors and prevent non-specific antibody binding, incubated for 15 minutes at 4°C in the dark and stained with different combinations of fluorescein isothiocyanate (FITC), PE, PE-Cy5, APC and PE-Cy7 and APC-Cy7-conjugated mAb and their appropriate isotype controls. Intracellular staining for Foxp3 was performed according to the instructions of the manufacturer eBiosciences (San Diego, CA, USA). The samples were analyzed on a FACSCanto II (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) using FACSDiva software v.6.1.2 (BD Bioscience) and FlowJo software v 7.1.2 (Tree Star). Lymphocytes were gated according to the Forward Scatter and Side Scatter. In addition gated CD4+ cells were analyzed for the expression of CD25 and Foxp3. Proportions of CD25+Foxp3+ and CD25-Foxp3+ cells within the gated CD4+ cells are shown. Absolute numbers of cells were calculated from whole blood counts obtained from routine laboratory testing.
Urine analysis
In patients with renal involvement, the extent of proteinuria was determined by measuring total protein in a 24-h urine collection specimen. Cells from 24-h urine samples from three patients were isolated by density gradient centrifugation. Cells were subjected to phenotypic analysis as described above.
Statistical analysis
Values are shown throughout the manuscript as mean ± standard error of the mean (SEM), unless stated otherwise. Proportions of lymphocyte subpopulations were compared using the Student t-test for normally distributed populations and if the variables were not normally distributed we used the Kruskal-Wallis test. Relationships between separate groups of data were examined using the Pearson correlation coefficient and Spearman rank correlation test. A P-value equal or less than 0.05 was considered significant in all statistical tests. All statistical analyses were performed using GraphPad Prism (Graph Pad Prism 4.0 by Graph Pad software Inc.) and SPSS (SPSS 12.0 by SPSS software Inc.).
Discussion
In this study we analyzed the recently identified population of CD4
+CD25
-Foxp3
+ T cells in a cohort of SLE patients. Extended phenotypic analysis based on the expression of the Ikaros transcription factor family member Helios support our previous assumption [
18] that the majority of CD4
+CD25
-Foxp3
+ T cells resemble Treg. We further described a connection between the disease activity and peripheral blood CD4
+CD25
-Foxp3
+ T cells. In addition we showed for the first time that the increment of CD4
+CD25
-Foxp3
+ T cells is linked to a specific organ manifestation, namely to renal involvement. A correlation between CD4
+CD25
-Foxp3
+ cells and the extent of proteinuria further support the idea of CD4
+CD25
-Foxp3
+ cells as a marker to recognize and monitor patients with renal involvement.
Initial studies regarding frequencies of Treg in the peripheral blood of SLE patients have generated controversial results. Most groups, including our own, have described decreased proportions of CD4
+CD25
high Treg in SLE patients as compared to healthy controls and observed an inverse correlation of Treg numbers with clinical disease activity [
7,
19,
29‐
31]. However, other studies reported unaltered proportions or even increased proportions of Treg in SLE patients and a positive correlation with disease activity [
17,
19,
32‐
34].
Among the overall population of CD4
+CD25
+ Treg we have recently identified a population of CD4
+Foxp3
+ T cells that lack the expression of CD25. Proportions of CD4
+CD25
-Foxp3
+ T cells were on average five-fold higher in SLE patients as compared to HC [
18]. Meanwhile this finding has been confirmed by several other groups who also described increased proportions of CD4
+Foxp3
+ T cells that are CD25-negative or express only low levels of CD25 in SLE patients [
19,
21,
23,
34].
Helios has recently been identified as a marker to distinguish thymic-derived Treg from Treg that are induced in the periphery [
15,
16]. Within this study we observed lower proportions of Helios
+ cells among CD4
+CD25
-Foxp3
+ T cells as compared to CD4
+CD25
+Foxp3
+ T cells, suggesting that CD4
+CD25
-Foxp3
+ T cells might reflect a combination of thymic-derived and peripherally induced Treg cells. In addition we found significant correlation between CD4
+CD25
-Foxp3
+ T cells and CD4
+CD25
+Foxp3
+ bona fide Treg cells, but not with CD4
+CD25
+Foxp3
- T cells, representing activated T cells. This together suggests that CD4
+CD25
-Foxp3
+ T cells represent regulatory T cells rather then activated T cells.
Concerning the influence of the disease activity on proportions of CD4
+CD25
-Foxp3
+ T cells, however, certain discrepancies exist. We therefore addressed this question in more detail in an extended cohort of SLE patients. Thereby we were able to describe a correlation between proportions of CD4
+CD25
-Foxp3
+ T cells and disease activity in SLE patients using three different disease activity scores. Moreover, we observed significant correlation between CD4
+CD25
-Foxp3
+ T cells with levels of anti-dsDNA antibodies and complement levels. In line with our data Lin
et al. reported a significant increase of CD4
+Foxp3
+ T cells in patients with active SLE, determined by the SLEDAI score, as compared to patients with inactive SLE or HC. In contrast, however, they observed no correlation between CD4
+CD25
-Foxp3
+ T cells and disease activity, the complement levels and concentrations of anti-dsDNA antibodies [
34]. Suen
et al. also described increased proportions of CD25
lowTreg and CD25
-Treg in patients with active and inactive SLE as compared to HC. Although they focused mainly on the ratio between different Treg subsets and effector T cells, they observed increased proportions of CD25
-Treg in patients with low complement levels. Similar observations of increased numbers of CD4
+CD25
-Foxp3
+ T cells in patients with active as compared to inactive SLE or HC were further made by Zhang
et al. Moreover they also reported direct correlation between proportions of CD4
+CD25
-Foxp3
+ T cells and concentrations of antibodies against dsDNA, whereas no correlation was observed for the C3 levels and the SLEDAI score [
19]. These discrepancies might be on the one hand explained by different definitions of patients with active and inactive SLE. On the other hand differences might exist in the composition of the study populations, in particular in regard to different organ manifestation as discussed in more detail below.
To further exclude a treatment effect on the expression of CD25 on T cells we compared proportions of CD4+CD25-Foxp3+ T cells in SLE patients with different treatment regimens. Thereby we only observed increased proportions in patients treated with cyclophosphamide. Further analysis, however, revealed that all cyclophosphamide-treated patients also suffered from active nephritis. In addition we observed no significant differences for absolute cell numbers of CD4+CD25-Foxp3+ T cells in SLE patients with other treatment regimens.
Significant correlation was found between CD4
+CD25
-Foxp3
+ T cells and the daily cortisone dose. Patients with active SLE, however, are usually treated with a higher cortisone dose and in fact this was confirmed by a significant correlation between the daily cortisone dose and the SLEDAI score. In addition we were able to follow one newly diagnosed SLE patient with high disease activity, and skin-, blood- and joint-manifestation, who was treated with prednisone. This resulted in gradually reduced disease activity without a concomitant substantial effect on proportions of CD4
+CD25
-Foxp3
+ T cells. Likewise, also other studies did not describe a correlation between Treg and the daily cortisone dose in SLE patients [
17,
23] except for Zhang
et al. who observed decreased proportions of CD4
+CD25
-Foxp3
+ T cells after treatment of active SLE with cortisone [
19]. An explanation for this discrepancy might be the different treatment regimen, as all patients in the study by Zhang
et al. also received cyclophosphamide at the same time. On the other hand, differences in the organ manifestations might contribute to this discrepancy.
As a systemic autoimmune disease SLE can affect almost any organ system and most patients display multi-organ involvement with unpredictable exacerbations and remissions with protean clinical manifestations. The most common organs that are involved are the skin, the musculoskeletal system and the kidneys. Organ systems may be involved singly or in any combination. Until now the association of a certain organ manifestation with the presence of different T cell subsets has not been investigated so far. As we also observed an increase of CD4+CD25-Foxp3+ T cells in cyclophosphamide-treated patients who suffered from active nephritis we compared CD4+CD25-Foxp3+ T cells in patients with active and no active organ involvement. Interestingly, patients with renal involvement, especially patients with active nephritis, displayed increased proportions of CD4+CD25-Foxp3+ T cells, suggesting that the observed increase was mainly associated with renal organ involvement. We also observed elevated numbers in patients with active blood involvement. This observation, however, was mainly due to an overlap in the organ manifestation since most of the patients with hematologic manifestations also suffered from renal involvement. When we compared absolute cell numbers of CD4+CD25-Foxp3+ T cells, we only observed a significant difference in patients with renal involvement, but not in patients with hematologic manifestation. Furthermore CD4+CD25-Foxp3+ T cells were also detected in the urine sediment of patients with active nephritis. In addition we also observed significant correlation between the CD4+CD25-Foxp3+ T cells and the extent of proteinuria, reflecting the disease activity in patients with active nephritis. Longitudinal analysis of patients with active nephritis revealed a decline in CD4+CD25-Foxp3+ T cells under cyclophosphamide treatment in parallel to a drop in the extent of proteinuria and the disease activity. The parallel decrease and increase of CD4+CD25-Foxp3+ T cells with the disease activity in SLE patients with renal manifestation might reflect the potential of CD4+CD25-Foxp3+ T cells as a biomarker to diagnose and monitor SLE patients with active kidney involvement.
One of the key questions remains whether CD4+CD25-Foxp3+ T cells actually represent a beneficial counter-mechanism against autoimmunity, or on the contrary, if these cells are part of the damaging auto-immunological machinery of SLE. In fact Treg display a certain plasticity, in particular under inflammatory conditions, and might even transdifferentiate into pathogenic effector T cells. However, until an activation-independent surface marker molecule is found for Treg, this question cannot be answered to date. On the other hand, irrespective of their functional capacity, CD4+CD25-Foxp3+ T cells might be of interest in SLE patients as a marker of disease activity, organ manifestation or treatment response.
In conclusion we were able to demonstrate that this newly described cell population of CD4+CD25-Foxp3+ T cells is clearly influenced by the disease activity and that an increase of this cell population is mainly observed in patients with renal involvement. The correlation with the extent of proteinuria suggests that the assessment of CD4+CD25-Foxp3+ cells can be used as a tool to recognize and monitor SLE patients with renal involvement.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MB: conception and design, data collection and analysis, manuscript writing, critical revision and final approval of the manuscript. LG: data collection and analysis, critical revision and final approval of the manuscript. SB: data collection and analysis, critical revision and final approval of the manuscript. TK: data collection and analysis, critical revision and final approval of the manuscript. CWS: data collection and analysis, critical revision and final approval of the manuscript. GS: conception and design, financial support, critical revision and final approval of the manuscript. JSS: conception and design, manuscript writing, financial support, critical revision and final approval of the manuscript. CS: conception and design, manuscript writing, critical revision and final approval of the manuscript. All authors read and approved the final manuscript.