You are here

  • Home
  • Archive
  • Volume 96, Issue 10
  • Low responder T cell susceptibility to the suppressive function of regulatory T cells in patients with dilated cardiomyopathy

Article Text

Article menu

Download PDFPDF

Heart failure and cardiomyopathy
Low responder T cell susceptibility to the suppressive function of regulatory T cells in patients with dilated cardiomyopathy
  1. Hongxia Tang1,a,
  2. Yucheng Zhong1,a,
  3. Yuntao Zhu1,
  4. Fang Zhao2,
  5. Xiaoxue Cui1,
  6. Zhaohui Wang1
  1. 1Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
  2. 2Department of Cardiology, Zhongnan Hospital of Wuhan University, Wuhan, China
  1. Correspondence to Professor Zhaohui Wang, Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, 430022, China; sgwangtjm{at}163.com

Abstract

Objective The pathogenesis of dilated cardiomyopathy (DCM) is closely connected with dysfunction of the autoimmune system, and CD4+CD25highCD127low/− regulatory T (Treg) cells have a vital role in maintaining self-tolerance. In this study, we compared the frequency and regulatory function of Treg cells between DCM patients and normal controls.

Methods and Results The frequencies of CD4+CD25+ T cells in DCM patients were statistically decreased compared with normal controls (p<0.05) by flow cytometry, and the levels of FOXP3 mRNA and protein expression in PBMCs (peripheral blood mononuclear cells) of DCM patients were lower than those of normal controls (p<0.01), using real-time RT-PCR assay and western blot. Notably, the suppressive capacity of CD4+CD25highCD127low/− regulatory T cells of DCM patients acting on autologous CD4+CD25− responder T (Tresp) cells seemed to be partially impaired (43.83±3.19% suppression versus 63.17±3.66% in normal controls, p=0.01). Surprisingly, Treg cells from DCM patients efficiently suppressed the proliferation of Tresp cells from normal subjects to the similar level as Treg cells from normal subjects on autologous Tresp cells (p=0.286), whereas Treg cells of normal subjects poorly inhibited the proliferation of Tresp cells from DCM patients.

Conclusion The defective capacity of Treg cells suppressing autologous Tresp cells is attributed to the increasing resistance of Tresp cells to inhibition of Treg cells in DCM patients. Therefore, strategies to improve the susceptibility of Tresp cells to Treg cell-mediated suppression might benefit DCM patients.

  • T-lymphocytes, regulatory
  • responder T cell
  • cardiomyopathy, dilated
  • FOXP3 protein, human
  • suppressive function

https://doi.org/10.1136/hrt.2009.184945

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    Introduction

    It is well established that the cardiovascular system is vulnerable to immune system modulation, especially viral myocarditis and its progression to dilated cardiomyopathy (DCM). DCM mostly resulting from viral myocarditis1 is characterised by an enlarged left ventricle or both enlarged ventricles with reduced contractility in the absence of abnormal loading conditions or ischaemic heart disease and evolves into heart failure.2 Although the underlying pathogenic mechanisms of the transition from viral myocarditis progressing to DCM have remained elusive, accumulating evidence from clinical observations and insights from animal models have proved that an autoimmune response is involved in the pathogenesis of DCM.3–7 Consequently, controlling autoimmunity might effectively help to attenuate the progression of DCM.

    The immune system can discriminate between self and non-self by central and peripheral tolerance mechanisms to maintain immunological homeostasis.8 The former involves deletion of autoreactive T cells in the thymus at an early stage of development (negative selection).9 However, negative selection is not absolute and even a considerable proportion of autoreactive T cells exist in the peripheral circulation in humans.10 Therefore, several mechanisms of peripheral tolerance, including anergy, ignorance and dominant suppression by Treg cells, are essential for clearing these autoreactive T cells.10 Treg cells, in particular, have an indispensable role in the tolerance mechanisms. For example, in animal models, it has been shown that depletion of the T cell subset alone was sufficient to cause autoimmune disease, whereas their reconstitution could efficiently suppress the activation and proliferation of autoreactive T cells to prevent organ-specific autoimmune diseases.11 12 Moreover, the Treg cell subset was capable of directly suppressing the proliferation and blasting of B cells, depletion of this subset resulted in a deregulated humoral response, which culminated in the production of autoantibodies.13 Treg cells appear to be one of the cornerstones of peripheral self-tolerance. Awareness is increasing that deficiency or dysfunction of CD4+CD25+FOXP3+ Treg cells may predispose to autoimmune diseases.14 For instance, CD4+CD25+Foxp3+ T cell-induced by H310A1 virus can effectively abrogate autoimmunity to prevent the development of DCM in tumour necrosis factor-α (TNF-α) transgenic mice.3 Therefore, we hypothesised that a quantitative and/or a qualitative abnormality of the Treg cells could contribute to the progression of autoimmunity existing in human DCM. In addition, it should been noted that FOXP3, a new member of the forkhead/winged-helix family of transcriptional regulators, is not only required for the generation and function of regulatory T cells, but also is currently one of the most specific markers for Treg cells.15 16 Nevertheless, FOXP3 cannot be used to isolate living Treg cells because of its intracellular expression. In recently published studies, CD25high was used to investigate the nature of regulatory T cells,17 18 but CD25 can also be expressed on activated non-regulatory T cells,19 Fortunately, the IL-7 receptor-chain (CD127) expression on CD4+ Treg cell surface has been detected to be low and is inversely correlated with FOXP3 expression on human CD4+ Treg cells, so low expression of CD127 combined with high expression of CD25 facilitates the isolation and purification of Treg populations among CD4+CD25+ T cells.20 21 Consequently, we sorted Treg cells according to the CD4+CD25highCD127low/− phenotype.

    In this paper, our data showed that in DCM patients, the percentages of CD4+CD25+ T cells within CD4+ T cells were reduced and FOXP3 mRNA and protein levels were obviously decreased in comparison with normal controls. Extraordinarily, CD4+CD25highCD127low/− Treg cells in DCM patients didn't show an intrinsic impaired regulatory function, but the sensitivity of Tresp cells from DCM patients to the suppressor activity of Treg cells was defective in the proliferation assay. Therefore, strategies to improve the susceptibility of Tresp cells to Treg cell-mediated inhibition might benefit DCM patients.

    Materials and methods

    Study subjects

    Twenty-five patients (mean age 53 years, range 26–75 years, eight female, 17 male) with a diagnosis of DCM without detectable aetiology were enrolled in this study. The diagnosis of DCM based on the criteria of WHO/ISFC of 1995. The exclusion criteria were (1) other autoimmune diseases; (2) tumour; (3) pregnancy; (4) endocrine disease; (5) serious infection recently; (6) receiving immunosuppressive agents (glucocorticosteroid). Endomyocardial biopsy was not studied in the patients, but most of them had a history of viral myocarditis. In this study, we chose cardiac autoantibody-positive patients as far as possible to further support the diagnosis of DCM based on previous studies, which showed that the use of synthetic peptides representing main antigenic determinants of autoantigens will certainly enhance the diagnostic repertoires in DCM.22 23 All patients were treated with digoxin, furosemide, various nitrate preparations and ACEI (angiotensin-converting enzyme inhibitor). Cardiological characteristics are listed in table 1 in detail. Normal controls were healthy volunteers (mean age 34 years, range 21–61 years, eight female, 17 male) who had no history of autoimmune disease and AHA (anti-heart autoantibodies) (including anti-ANT (adenine nucleotide (ADP/ATP) translocator), MHC (myosin heavy chain), β1 (β1-adrenergic receptor) and M2 (muscarinic receptors-2)) antibodies were negative in their sera. Although AHA may exist in the peripheral blood of healthy people, we excluded people who were cardiac antibody-positive in case of asymptomatic viral myocarditis. All patients and normal controls gave written informed consent prior to the study. Ethical approval for this study was granted by the ethics committee of Tongji Medical College of Huazhong University of Science and Technology. The investigation conforms with the principles outlined in the Declaration of Helsinki.

    View this table:
    Table 1

    Cardiological characteristics of patients with dilated cardiomyopathy

    Peptide synthesis

    Selected amino-acid sequences were synthesised with the fluorenylmethoxycarbonyl/t-butyl-based solid-phase peptide chemistry method by using a PSSM-8 automated peptide synthesiser (Shimadzu, Japan). The purities of these seven synthetic peptides examined by high liquid chromatography were up to 95%. The amino acid sequences of seven peptides are shown in table 2.

    View this table:
    Table 2

    Sequences of seven peptides

    Enzyme-linked immunosorbent assay (ELISA)

    The level of AHA was determined by ELISA. Microtitre plates coated with purified peptides 50 μl per well of 20 μg/ml in a 0.5 M alkaline buffer containing sodium carbonate and sodium acid carbonate (pH 9.6) were incubated overnight at 4°C, followed by blocking with PBS containing 10% BSA and three washings with PBS containing 0.05% Tween-20. Then serum samples were added to the plates at 1 μl /well in suitable dilution (1:100) and incubated for 60 minutes at 37°C. After additional washing, a horseradish peroxidase-labelled goat anti-human IgG was added at a concentration of 1/15000 (Gibco, USA) to react for 60 minutes at 37°C. Then the plates were washed and developed for 15 minutes with the substrate solution (0.01% H2O2 and 0.1% 3,3′,5,5′-tetramethyl benzidine), and the reaction then stopped by adding 2 M H2SO4. Optical density (OD) was read at 450 nm with an automated ELISA reader.

    Cell isolation

    Peripheral blood mononuclear cells (PBMCs) were isolated from heparinised peripheral blood by Ficoll-Hypaque density gradient centrifugation and either used for flow cytometry, or further isolated using the CD4+CD25+CD127dim/− Regulatory T Cell Isolation Kit human (Miltenyi Biotec Bergisch, Gladbach, Germany) according to the manufacturer's protocol. CD4+CD127low/− T cells were isolated from PBMCs using a CD4+ T cell negative isolation kit (Miltenyi Biotec). The CD25+ fraction (CD4+CD25highCD127low/− T cells) was further enriched by positive selection using CD25 microbeads. CD4+CD25highCD127low/− T cells were purified to a purity of 90-95% using a Vantage fluorescence-activated cell sorter (FACS- Calibur; BD Biosciences, USA).

    Cell culture medium

    Cells were grown in RPMI 1640 (Gibco, USA) with 10% fetal calf serum (Gibco), 25 mM Hepes, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (BioWhittaker, USA), 0.05 mM of non-essential amino acids (Gibco, USA), in 96-well microplates (CoStar, USA).

    Flow cytometric analysis

    PBMCs were stained with a PE-conjugated anti-CD4 mAb and FITC-anti-CD25 (BD Biosciences). PBMCs were incubated for 20 minutes at 4°C in the dark. After washing with PBS, cells were analysed by flow cytometry. Flow cytometry data were collected on a FACS-Calibur (BD) and analysed using Cell Quest software (BD).

    Western blot

    A total of 2×106 PBMCs from DCM patients or normal controls were lysed in RIPA buffer. Protein levels were checked with a BCA kit (Pierce Biotechnology Inc). After protein was heated at 95°C for 5 minutes, 35 mg of protein was loaded into each lane of a 9% SDS-PAGE gel and transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). The blot was incubated with 1:1000 primary antibody (Affinity Purified anti-human FOXP3 (eBioscience, USA)) overnight at 4°C, followed by incubation with the secondary horseradish peroxidase-conjugated antibody (1:5000) (Pierce, USA) for 2 hours at room temperature. Then the blot was detected with the ECL detection kit (Pierce, America) and exposed to a Koda x-ray film. After stripping, the membrane was reprobed with an anti-actin Ab (eBioscience, USA). Band intensities were quantified with Quantity One (Bio-RAD, USA). Integrated density values (IDV) for the test (FOXP3) and control (β-actin) bands were obtained and expressed as their ratio.

    Semiquantitative analysis of FOXP3 mRNA by real-time RT-PCR

    Total RNA was extracted with Trizol reagent (Invitrogen, USA) from PBMCs of DCM patients and normal controls and reverse transcribed to cDNA using RNA PCR Kit (Takara Biotechnology, Dalian, China), transcription reagents using M-MLV reverse transcriptase and oligo (dT) 12-18 (Invitrogen), according to the manufacturer's instructions. The following primers were used to amplify FOXP3 and β-actin: FOXP3 forward: 5′-CTACGCCACGCTCATCCGCT-3′, reverse: 5′-GGTCCACACAGCCCCCTTCT-3′. β-actin forward: 5′-CGAAACTACCTTCAACTCCATCA-3′, reverse: 5′-CGGACTCGTCATACTCCTGCT-3′. Gene expression was measured in real-time PCR with the ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using primers and Quanti Tect SYBR green PCR Kit (Biotium, USA). Melting curves established the purity of the amplified band after 45 cycles of 30 seconds at 94°C, 30 seconds at 57°C and 30 seconds at 72°C. Relative FOXP3 mRNA expression level was calculated using the comparative Ct method formula 2−∆∆ct. Data are presented and normalised to β-actin.

    Proliferation assay

    We isolated CD4+CD25− (responders) and CD4+CD25highCD127low/− (suppressors) T cells from PBMCs in eight DCM patients and eight normal controls by CD4 negative selection followed by CD25 positive selection. Then the function of CD4+CD25highCD127low/− Treg cells was assessed under different conditions: (1) CD4+CD25- T cells (responders) (1×104 cells/well) alone; (2) CD4+CD25highCD127low/− Treg cells (1×104 cells/well) alone; (3) CD4+CD25− T cells were co-cultured with autologous CD4+CD25highCD127low/− Treg cells at different ratios (responder/suppressor ratios: 1:1, 1:1/2, 1:1/4, and 1:1/8); (4) crossover culture, CD4+CD25highCD127low/− Treg cells from DCM patients were co-cultured with CD4+CD25− T cells from normal controls at a 1:1 ratio, CD4+CD25highCD127low/− Treg cells from normal controls were co-cultured with CD4+CD25− T cells from DCM patients at 1:1 ratio. T cells were incubated in 1640 medium with 10% FBS in 96-well microplates coated with anti-CD3 (Clone: OKT3; eBioscience) at 1.5 μg/ml and soluble anti-CD28 (clone 28.6 eBioscience) at 2 μg/ml. All cells were cultured in a final volume of 150 μl. Cultures were incubated for 72 hours at 37°C in a humidified, 5% CO2-containing incubator. After 3 days of incubation, cell proliferation was evaluated by adding 0.5 μCi 3H-thymidine directly to the culture wells and incubating them for 16 hours at 37°C, then the cells were harvested; the counts per minute (cpm)/well was determined by scintillation counting (PerkinElmer). Percentage suppression was determined as 1 − (cpm incorporated in the co-culture)/cpm of responder population alone ×100%.

    Statistical analysis

    Data were expressed as the mean±SEM. The Mann-Whitney U test was used to detect significant differences between the two groups. A statistical value of <0.05 was considered statistically significant. For statistical analyses, SPSS 13.0 software was used.

    Results

    The frequencies of CD4+CD25+ T cells in peripheral blood of DCM patients were lower than those in normal controls.

    In this study, we assessed the percentages of CD25 expression on CD4+ T cells and CD4+ T subset within PBMCs from DCM patients and normal controls by flow cytometry (figure 1). We found that there was no significant difference in the percentage of CD4+ T cells in PBMCs between DCM patients and normal controls (p>0.05) (table 3). However, the frequency of CD4+CD25+ T cells in DCM patients (mean±SD, 11.3±4.8%, n=25) was statistically reduced compared with normal controls (p<0.05) (table 3).

    Figure 1
    Figure 1

    The percentages of CD25 expression on gated CD4+ T cells and CD4+ T subset within PBMCs from human were assessed by flow cytometry. (A) Lymphocytes were identified by their forward and side-scatter properties. (B) PBMCs were stained with CD4-PE and expression of gated CD4+ T cells was presented as the positive cells. (C) PBMCs were double-stained for CD4+ and CD25+ using PE-anti-CD4 and FITC-anti-CD25 and the frequencies of CD4+CD25+ T cells were assessed.

    View this table:
    Table 3

    The percentages of CD25 expression on gated CD4+ T cells and CD4+ T subset within PBMCs from DCM patients and normal controls

    FOXP3 protein and mRNA expression levels from PBMCs of DCM patients were remarkably reduced compared with normal controls

    FOXP3 is indispensable for the generation and development of Treg cells.15 16 Furthermore, the mutations of Foxp3 protein resulted in a fatal autoimmune disorder regarded as the immune dysregulation, polyendocrinopathy, enteropathy and X-linked (IPEX) syndrome.25 26 Hence it is very important to quantitate the expression of FOXP3 in DCM patients. Real-time RT-PCR analysis indicated that FOXP3 mRNA levels were remarkably reduced in PBMCs from DCM patients (mean±SEM, 20.8±5.9, n=25) when compared with normal controls (139±39.07, n=25; Mann-Whitney U test, p<0.001, figure 2A). Notably, FOXP3 genes might be regulated at the post-transcriptional level. We examined Foxp3 protein expression by western blot analysis. Foxp3 protein expression was also obviously decreased in DCM patients (n=25, mean±SEM, 37±2.8%) in comparison with control controls (n=25, 60±4.5%, p=0.001, figure 2B,C).

    Figure 2
    Figure 2

    Comparative analysis of the levels of FOXP3 mRNA and protein expression in PBMCs derived from DCM (n=25) and normal controls (n=25) by real-time RT-PCR and western blot. β-actin was used as a reference gene. Normalised FOXP3 mRNA and protein abundance were determined from the ratio of FOXP3 mRNA and protein to β-actin mRNA and protein expression, respectively. (A) Relative mRNA expression of FOXP3 in PBMCs from DCM patients was remarkably reduced compared with normal controls (p<0.001). (B) Lanes 2 and 4 represent FOXP3 protein abundance in DCM patients, while the lanes 1, 3 and 5 represent that in normal controls. β-actin was used as control protein. (C) All western blot lanes were analysed by a Quantity One (Bio-RAD). Integrated density values (IDV) for FOXP3 and control (β-actin) lanes were obtained and expressed as their ratio. The relative level of FOXP3 protein expression was decreased in DCM patients compared with normal controls (p=0.001). Data are expressed as the mean±SEM. Each bar represents an average of the data from DCM patients or normal controls. Error bars represent SEM.

    The defective suppressive capacity of CD4+CD25highCD127low/− Treg cells in co-culture with autologous CD4+CD25− T cells in DCM patients compared to normal controls was the result of the low sensitivity of Tresp cells to the activity of Treg cells.

    To quantify Treg cell function based on the suppressive effect of Treg cells on Tresp cell proliferation, we separated CD4+CD25highCD127low/− Treg cells and CD4+CD25− T cells from eight DCM patients and eight normal controls by the CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit human, then CD4+CD25highCD127low/− Treg cells (suppressors) were co-cultured with autologous CD4+CD25− T cells (responders) at different ratios (responder/suppressor ratios: 1:1, 1:1/2, 1:1/4 and 1:1/8). Because Treg cells can exert their suppressor effect following activation via their TCR, anti-CD3 and anti-CD28 mAbs, which cannot abrogate the anergic state and regulatory properties of Treg cells,27 were used to stimulate CD4+CD25highCD127low/− Treg cells in the experiment. Figure 3A showed that the proliferation of CD4+CD25highCD127low/− Treg cells was minimal, which suggested that they were anergic to simulation via the TCR consistent with CD4+CD25high T cell in other studies.27 Furthermore, the proliferative response of CD4+CD25− T cells was drastically inhibited in the presence of CD4+CD25highCD127low/− Treg cells. In the meanwhile, the regulatory capacity of Treg cells is in a dose-dependent manner, in that increasing the ratio of responder/suppressor T cells resulted in less suppression (figure 3B). Moreover, the percentages of inhibition proliferation of CD4+CD25- Tresp cells by autologous CD4+CD25highCD127low/− Treg cells at different ratios from DCM patients were lower than those from normal controls, especially at a Treg:Tresp ratio of 1:1 (figure 3B, mean±SEM, 43.83±3.19% suppression in DCM patients (n=8) vs 63.17±3.66% in normal controls (n=8); Mann-Whitney U test, p=0.01), it seemed that Treg cells from DCM patients were significantly less potent than those from normal controls in suppressing the proliferation of Tresp cells.

    Figure 3
    Figure 3

    Comparison of the regulatory potent of CD4+CD25highCD127low/− Treg cells in DCM patients and normal controls by suppression of proliferation assays. All cells in experiments were stimulated with plate-bound anti-CD3 at 1.5 μg/ml and anti-CD28 at 2 μg/ml. After 3 days of incubation, proliferation was measured by adding 3H-thymidine directly. (A) Comparison of the proliferation of CD4+CD25highCD127low/− T cells in DCM patients (n = 8) and normal controls (n=8) normalised against the proliferation of the CD4+CD25− T cell subset from normal controls. The proliferation of CD4+CD25highCD127low/− Treg cell subset (grey bars) is minimal, indicating it is anergic to stimulation. Bars show the mean and SEM. (B) Sorted CD4+CD25highCD127low/− T cells from DCM patients (black squares, n=8) or normal controls (black oblique squares, n=8) were co-cultured with autologous CD4+CD25- T cells at different ratios (responder/suppressor ratios: 1:1, 1:1/2, 1:1/4 and 1:1/8). The percentage suppression of CD4+CD25− T cell (responder T cell) proliferation by CD4+CD25highCD127low/− Treg cells was determined as 1 − (proliferation of co-culture/proliferation of responder population alone) ×100%. Percentages inhibition of proliferation of CD4+CD25- T cells by CD4+CD25highCD127low/− Treg cell was decreased with the increasing ratio of Tresp/Treg. Results are the mean±SEM of eight separate experiments. (C) Sorted CD4+CD25highCD127low/− Treg cell (1×104/well) from DCM patients suppressed proliferation of CD4+CD25− T cells derived from either the autologous individuals (n=8) or normal controls (n=8) at 1:1 ratio, while CD4+CD25highCD127low/− Treg cells (1×104/well) isolated from normal controls were co-cultured with CD4+CD25− T cells from either autologous individuals (n=8) or DCM patients (n=8) at 1:1 ratio. The results represent the mean±SEM. Significant differences were calculated using the non-parametric Mann-Whitney U test.

    A note of caution, it was also important to examine whether the defective regulatory function in the co-culture systems of cells from DCM patients was due to a decrease in CD4+CD25highCD127low/− Treg cell function or an increase in the resistance of activated CD4+CD25− T cell to inhibition. Thus, a crossover experiment was performed based on the antigen non-specific suppressive function of Treg cells.27 28 In striking contrast, there was no significant difference in the percentage inhibition of Tresp cells proliferation from normal controls between CD4+CD25highCD127low/− Treg cells of DCM patients and those of normal controls (55%±10.53% vs 63.17±3.66%, Mann-Whitney U test, p=0.286), whereas the proliferation of Tresp cells from DCM patients could not efficiently be suppressed by both autologous Treg cells (mean±SEM, 44±3.19%, n=8) and Treg cells of normal controls (41.25±7.22%, n=8; p=0.748). Furthermore, the regulatory potency of CD4+CD25highCD127low/− Treg cells from normal controls suppressing the proliferation of Tresp cells from DCM patients was less than that of them acting on autologus Tresp cells (41.25±7.22% vs 63.17±3.66%, p=0.025) in agreement with a recently published study in patients with active systemic lupus erythematosus29 (as shown in figure 3C). Based on the experimental results, it can be concluded that the regulatory function of CD4+CD25highCD127low/− Treg cells was not defective, but the sensitivity of Tresp cells to the suppression of Treg cells was impaired in DCM patients.

    Discussion

    Abundant experimental data have now confirmed that the pathogenesis of autoimmune diseases is closely associated with the disorder of immune tolerance,14 30 which includes ‘passive’ and ‘active’ mechanisms, because these ‘passive’ mechanisms for self-tolerance may not be sufficient to completely purge autoreactive T cells. Therefore, the 'active' mechanism of immune suppression in which there is a unique population of regulatory T cells seems to be very important to inhibit both the induction and effector function of autoreactive T cells, thereby preventing autoimmune diseases.11 12 30 In a mouse model, CD4+CD25+ T cells from normal mice were highly effective in inhibiting a spectrum of organ-specific autoimmunity that was induced in susceptible mouse strains by thymectomising pups at day 3 of life.11 12 In humans, it was demonstrated that there was a quantitative or/and a qualitative abnormality of Treg cells in patients with autoimmune diseases, such as multiple sclerosis (MS)17 and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).18 It is evident that the autoimmune mechanism has a key role in the pathogenesis of DCM on the basis of the demonstration of mononuclear cell infiltrates and autoantibodies against the myocardium.4–7 Most importantly, depletion of T cells in TNF-α transgenic mice using monoclonal anti-CD3 or anti-CD4 antibody could significantly reduce heart size.3 From the experiment results, it is obvious that autoaggressive CD4+ T cell response and humoral autoimmunity play an important pathogenic part in DCM. Therefore, we deduced that Treg cells failed to efficiently inhibit autoimmunity in DCM for a decrease in the number or/and suppressive function. In this paper, our data showed that there was a significant decrease in the frequencies of Treg cells in peripheral blood of DCM patients by flow cytometry based on their characteristic CD4+CD25+ membrane phenotype, but the percentage of CD4+ T cells in PBMCs was similar between DCM patients and normal controls, in accordance with the result of a study showing that the percentage of suppressor/cytotoxic T cells was low in idiopathic dilated cardiomyopathy compared with healthy controls and ischaemic heart disease.31 However, the frequency and number of Treg cells have been described as normal, low or high in other autoimmune diseases,17 18 29 32 so we conclude that many uncontrollable factors may result in the discrepant results. First, different autoimmune diseases maybe have different alteration of Treg cells in human peripheral blood. Second, it is worth noting that a heterogeneous population of CD4+CD25+ T cells includes low and moderate levels of CD25 lacking an immune suppressor function and high levels of CD25 exhibiting regulatory function,19 but there is no clear and stereotyped cut-off between high and intermediate CD25 expression in humans as the percentage of CD4+CD25highT cells within CD4+ T cells is not reliable. Therefore, we did not analyse the mean fluorescent intensity of the CD25+ population in both DCM patients and control subjects. Because the CD4+CD25+FOXP3+ T cell subset is ‘typical’ Treg, a limitation of our study was that we didn't use FOXP3 as an additional specific marker in the flow cytometry. Nevertheless, we analysed the FOXP3 expression level in PBMCs from DCM patients and normal controls. Foxp3 is most highly expressed in CD4+CD25+ Treg cells and is virtually undetectable in both stationary and activated of CD4+CD25− and CD8+ T cells and B cells.15 16 In this study, we reported that the levels of FOXP3 mRNA and protein expression from PBMCs of DCM patients were statistically reduced compared with normal controls. It is well accepted that Foxp3 governs the generation and function of regulatory T cells.15 16 Accordingly, we concluded that the declined expression level of FOXP3 led to a decrease in the number of Treg cells which may contribute to the progression of DCM. The conclusion was further supported by the observation that Foxp3 overexpression increased CD4+CD25+ T cell numbers,16 and it should be noted that the low levels of FOXP3 expression didn't result in a defect in the regulatory of Treg cells in this research. Some studies verified that mutations in Foxp3 led to a fatal T cell-mediated autoimmune disease in both mice and humans.25 26 We proposed that the declined expression of FOXP3 was not enough to affect the function of Treg cells, which suggests that a low expression of FOXP3 could keep the regulator function of Treg cells.

    Some earlier studies have already tested suppressor T lymphocyte function in patients with idiopathic dilated cardiomyopathy, but the data were discrepant; Fowles and Eckstein found that the suppressor cells function was defective in vitro.33 34 However, Lowry et al reported that there was no defect in suppressor T cell function in patients with congestive cardiomyopathy.35 Whatever the finding is, it is very important to define the suppressor cells according to the specific member markers for assessing the cell subset function. With the development of immunology, a number of cell surface phenotypes of Treg cell populations have been described over the past few years, strong evidence suggests that FOXP3 is a molecular marker unique to Treg cells.15 16 However, the intracellular expression of FOXP3 has hampered viable cell sorting for function assay, so it is crucial to take into consideration other member markers that discriminate Treg cells from activated T cells. Luckily, recent reports show that the IL-7 receptor (CD127) is inversely correlated with FOXP3 expression on human CD4+ Treg cells, and may be so far the best available marker for the identification of Treg cells.20 21 CD4+CD25highCD127low/− T cells exhibit potent suppressive activity and express more FOXP3 compared with CD4+CD25highT cells in humans.20 21 In the functional suppressor assays, as reported previously,27 CD4+CD25highCD127low/− Treg cells were anergic to simulation via the TCR and regulatory function was in dose-dependent manner. Notably, the suppressive function of CD4+CD25highCD127low/− Treg cells from DCM patients was significantly defective compared with that of CD4+CD25highCD127low/− Treg cells from normal controls in co-culture with autologous Tresp cells. However, in crossover experiments, we revealed that both Treg cells of DCM patients and normal controls could efficiently suppress the proliferation of Tresp cells from normal controls, whereas they could not well suppress the proliferation of Tresp cells from DCM patients, indicating that the suppressive function of CD4+CD25highCD127low/− Treg cells from DCM patients in fact is intact. The impaired sensitivity of Tresp cells can account for the defective suppressive function of CD4+CD25highCD127low/− Treg cell from DCM patient in co-culture systems. These results were in agreement with recently published studies in blood of patients with MS and active systemic lupus erythematosus.29 36 In contrast to the findings shown above, CD4+CD25highTreg cells derived from patients with autoimmune diseases exhibited defective regulatory function compared with those derived from healthy individuals.17 18 The contradictory results of the regulatory function of Treg cells might be interpreted by heterogeneity of the isolated Treg cell populations. For instance, CD127highT cells within the CD4+CD25high T cells might interfere with detecting suppressive function of CD4+C25high T cells in these patients with autoimmune diseases. Michel et al showed that CD4+CD25highCD127high T cells had significantly higher proliferation and produced higher levels of cytokines than CD4+CD25highCD127low T cells and CD4+CD25− T cells in MS patients,36 which can at least partly account for a decreased suppressive function of CD4+CD25high T cells in previous studies.

    Our data prompt us to research for the mechanisms of the resistance of Tresp to Treg suppressive function in future work. Some researchers put forward one possibility that the presence of an overwhelming inflammatory cascade of events may result in the resistance of Tresp cells. Thomas et al have described that Tresp cells derived from the central nervous system seemed to have unique property of secreting large amounts of IL-6 and TNF during experimental autoimmune encephalomyelitis (EAE), which override the ability of Treg cells to prevent autoimmune disease.37 O'Sullivan et al have shown that IL-1β induced the proliferation and cytokine production of Tresp cells, which was responsible for the resistance of the Tresp cells to Treg cell-mediated suppression in a diabetic mouse model.38 Here, we did not test the proinflammatory cytokines which led to responder cell resistance. However, it is worth noting that the proliferation of Tresp cells of DCM patients was more obvious than that of normal controls based on our experiment results.

    In summary, our study is the first to show that the frequency of CD4+CD25+ T cells from DCM patients is lower than that from normal controls, while PBMCs contained an equal proportion of CD4+ T cells in both groups. Moreover, the level of FOXP3 mRNA and protein of PBMCs in DCM patients was significantly decreased when compared with healthy controls. Significantly, we showed that the impaired suppressive function of CD4+CD25highCD127low/− T cells acting on the proliferation of autologous CD4+CD25− T cells is not a defect regulatory function of CD4+CD25highCD127low/− Treg cells, but a resistance of CD4+CD25− T cells to suppression in DCM patients. This observation provides a new dimension for further research on the mechanism of the Tresp cells refractory to inhibition of Treg cells, and it might prove to be beneficial for the development of a highly specific, effective immunotherapeutic approach for restoring systemic autoimmune diseases such as DCM. For example, it is possible that promoting the susceptibility of Tresp cells to Treg cell-mediated suppression by neutralising pro-inflammatory cytokines and increasing the number of Treg cells might attenuate the development of DCM.

    References

    1. ↵
      1. Kearney MT,
      2. Cotton JM,
      3. Richardson PJ,
      4. et al
      . Viral myocarditis and dilated cardiomyopathy: mechanisms, manifestations, and management. Postgrad Med J 2001;77:4–10.
      OpenUrlAbstract/FREE Full Text
    2. ↵
      1. Richardson P,
      2. McKenna W,
      3. Bristow M,
      4. et al
      . Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the definition and classification of cardiomyopathies. Circulation 1996;93:841–2.
      OpenUrlFREE Full Text
    3. ↵
      1. Huber SA,
      2. Feldman AM,
      3. Sartini D
      . Coxsackievirus B3 induces T regulatory cells, which inhibit cardiomyopathy in tumor necrosis factor-α transgenic mice. Circ Res 2006;99:1109–16.
      OpenUrlAbstract/FREE Full Text
    4. ↵
      1. Schulze K,
      2. Becker BF,
      3. Schauer R,
      4. et al
      . Antibodies to ADP-ATP carrier – an autoantigen in myocarditis and dilated cardiomyopathy – impair cardiac function. Circulation 1990;81:959–69.
      OpenUrlAbstract/FREE Full Text
    5. ↵
      1. Caforio AL,
      2. Grazzini M,
      3. Mann JM,
      4. et al
      . Identification of a- and b-cardiac myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 1992;85:1734–42.
      OpenUrlAbstract/FREE Full Text
    6. ↵
      1. Limas CJ,
      2. Goldenberg IF,
      3. Limas C
      . Autoantibodies against b-adrenoceptors in human idiopathic dilated cardiomyopathy. Circ Res 1989;64:97–103.
      OpenUrlAbstract/FREE Full Text
    7. ↵
      1. Fu LX,
      2. Magnusson Y,
      3. Bergh CH,
      4. et al
      . Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy. J Clin Invest 1993;91:1964–8.
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Parijs LV,
      2. Abbas AK
      . Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 1998;280:243–8.
      OpenUrlAbstract/FREE Full Text
    9. ↵
      1. Kappler JW,
      2. Roehm N,
      3. Marrack P
      . T cell tolerance by clonal elimination in the thymus. Cell 1987;49:273–80.
      OpenUrlCrossRefPubMedWeb of Science
    10. ↵
      1. Walker LS,
      2. Abbas AK
      . The enemy within: keeping self-reactive T cells at bay in the periphery. Nat Rev Immunol 2002;2:11–9.
      OpenUrlCrossRefPubMedWeb of Science
    11. ↵
      1. Payer ES,
      2. Amar AZ,
      3. Thornton AM,
      4. et al
      . CD4+CD25+T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol 1998;160:1212–18.
      OpenUrlAbstract/FREE Full Text
    12. ↵
      1. Shevach EM
      . Regulatory T cells in autoimmunity. Annu Rev Immunol 2000;18:423–49.
      OpenUrlCrossRefPubMedWeb of Science
    13. ↵
      1. Bystry RS,
      2. Aluvihare V,
      3. Welch KA,
      4. et al
      . B cells and professional APCs recruit regulatory T cells via CCL4. Nat Immunol 2001;2:1126–32.
      OpenUrlCrossRefPubMedWeb of Science
    14. ↵
      1. Sakaguchi S,
      2. Ono M,
      3. Setoguchi R,
      4. et al
      . Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 2006;212:8–27.
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      1. Fontenot JD,
      2. Gavin MA,
      3. Rudensky AY
      . Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003;4:330–6.
      OpenUrlCrossRefPubMedWeb of Science
    16. ↵
      1. Khattri R,
      2. Cox T,
      3. Yasayko SA,
      4. et al
      . An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol 2003;4:337–42.
      OpenUrlCrossRefPubMedWeb of Science
    17. ↵
      1. Viglietta V,
      2. Baecher-Allan C,
      3. Weiner HL,
      4. et al
      . Loss of functional suppression by CD4+CD25+regulatory T cells in patients with multiple sclerosis. J Exp Med 2004;199:971–9.
      OpenUrlAbstract/FREE Full Text
    18. ↵
      1. Chi LJ,
      2. Wang HB,
      3. Wang WZ
      . Impairment of circulating CD4+CD25+ regulatory cells in patients with chronic inflammatory demyelinating polyradiculoneuropathy. J Peripher Nerv Syst 2008;13:54–63.
      OpenUrlCrossRefPubMedWeb of Science
    19. ↵
      1. Allan CB,
      2. Brown JA,
      3. Freeman GJ,
      4. et al
      . CD4+CD25 high regulatory cells in human peripheral blood. J Immunol 2001;167:1245–53.
      OpenUrlAbstract/FREE Full Text
    20. ↵
      1. Gingeras BF,
      2. de St Groth C,
      3. Clayberger DM,
      4. et al
      . CD127 expression inversely correlates with FOXP3 and suppressive function of human CD4+ Treg cells. J Exp Med 2006;203:1701–11.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      1. Banham AH
      . Cell-surface IL-7 receptor expression facilitates the purification of FOXP3+regulatory T cells. Trends Immunol 2006;27:541–4.
      OpenUrlCrossRefPubMedWeb of Science
    22. ↵
      1. Schwimmbeck PL,
      2. Schwimmbeck NK,
      3. Schultheiss HP,
      4. et al
      . Mapping of antigenic determinants of the adenine-nucleotide translocator and coxsackie B3 virus with synthetic peptides: use for the diagnosis of viral heart disease. Clin Immunol Immunopathol 1993;68:135–40.
      OpenUrlCrossRefPubMedWeb of Science
    23. ↵
      1. Schwimmbeck PL,
      2. Bland NK,
      3. Schulthesis HP,
      4. et al
      . The possible value of synthetic peptides in the diagnosis and therapy of myocarditis and dilated cardiomyopathy. Eur Heart J 1991;12(Suppl D):76–80.
      OpenUrlAbstract/FREE Full Text
    24. ↵
      1. Magnusson Y,
      2. Marullo S,
      3. Hoyer S,
      4. et al
      . Mapping of a functional autoimmune epitope on the β1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy. J Clin Invest 1990;86:1658–63.
      OpenUrlCrossRefPubMedWeb of Science
    25. ↵
      1. Brunkow ME,
      2. Jeffery EW,
      3. Hjerrild KA,
      4. et al
      . Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 2001;27:68–73.
      OpenUrlCrossRefPubMedWeb of Science
    26. ↵
      1. Bennett CL,
      2. Christie J,
      3. Ramsdell F,
      4. et al
      . The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome is caused by mutations of FOXP3. Nat Genet 2001;27:20–1.
      OpenUrlCrossRefPubMedWeb of Science
    27. ↵
      1. Jonuleit H,
      2. Schmitt E,
      3. Stassen M,
      4. et al
      . Identification and Functional Characterization of Human CD4+CD25+ T Cells with Regulatory Properties Isolated from Peripheral Blood. J Exp Med 2001;193:1285–94.
      OpenUrlAbstract/FREE Full Text
    28. ↵
      1. Thornton AM,
      2. Shevach EM
      . Suppressor effector function of CD4+CD25+immunoregulatory T cells is antigen nonspecific. J Immunol 2000;164:183–90.
      OpenUrlAbstract/FREE Full Text
    29. ↵
      1. Venigalla RKCV,
      2. Tretter T,
      3. Krienke S,
      4. et al
      . Reduced CD4+, CD25-T cell sensitivity to the suppressive function of CD4+, CD25high, CD127-/low regulatory T cells in patients with active systemic lupus erythematosus. Arthritis Rheum 2008;58:2120–30.
      OpenUrlCrossRefPubMedWeb of Science
    30. ↵
      1. Sakaguchi S
      . Regulatory T cells: key controllers of immunologic self-tolerance. Cell 2000;101:455–8.
      OpenUrlCrossRefPubMedWeb of Science
    31. ↵
      1. Gerli R,
      2. Rambotti P,
      3. Spinozzi F,
      4. et al
      . Immunologic studies of peripheral blood from patients with idiopathic dilated cardiomyopathy. Am Heart J 1986;112:350–5.
      OpenUrlCrossRefPubMedWeb of Science
    32. ↵
      1. Nakamura K,
      2. Miki M,
      3. Mizoguchi Y,
      4. et al
      . Deficiency of regulatory T cells in children with autoimmune neutropenia. Br J Haematol 2009;145:642–7.
      OpenUrlCrossRefPubMed
    33. ↵
      1. Eckstein R,
      2. Mempel W,
      3. Bolte HD
      . Reduced suppressor cell activity in congestive cardiomyopathy and in myocarditis. Circulation 1982;65:1224–9.
      OpenUrlAbstract/FREE Full Text
    34. ↵
      1. Fowles RE,
      2. Bieber CP,
      3. Stinson EB
      . Defective in vitro suppressor cell function in idiopathic congestive cardiomyopathy. Circulation 1979;59:483–91.
      OpenUrlFREE Full Text
    35. ↵
      1. Lowry PJ,
      2. Gammage MD,
      3. Gentle TA,
      4. et al
      . Suppressor T lymphocyte function in patients with idiopathic congestive cardiomyopathy. Br Heart J 1987;57:458–61.
      OpenUrlAbstract/FREE Full Text
    36. ↵
      1. Michel L,
      2. Berthelot L,
      3. Pettré S,
      4. et al
      . Patients with relapsing-remitting multiple sclerosis have normal Treg function when cells expressing IL-7 receptor α-chain are excluded from the analysis. J Clin Invest 2008;118:3411–19.
      OpenUrlPubMedWeb of Science
    37. ↵
      1. Korn T,
      2. Reddy J,
      3. Gao W,
      4. et al
      . Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med 2007;13:423–31.
      OpenUrlCrossRefPubMedWeb of Science
    38. ↵
      1. O'Sullivan BJ,
      2. Thomas HE,
      3. Pai S,
      4. et al
      . IL-1β breaks tolerance through expansion of CD25+ effector T cells. J Immunol 2006;176:7278–87.
      OpenUrlAbstract/FREE Full Text
    View Abstract

    Footnotes

    • ↵a These authors contributed equally to this work.

    • Funding National Basic Research Program of China (973 Program)£°2007CB512000£»2007CB512005.

    • Competing interests None declared.

    • Patient consent Obtained.

    • Ethics approval This study was conducted with the approval of the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology.

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