Chinese Medical Journal 2008;121(18):1781-1786
Involvement of CD4+CD25+ regulatory T cells in the pathogenesis of polycythaemia vera

ZHAO Wen-bo,  LI Ying,  LIU Xin,  ZHANG Ling-yan,  WANG Xin

ZHAO Wen-bo (Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China)

LI Ying (Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China)

LIU Xin (Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China)

ZHANG Ling-yan (Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China)

WANG Xin (Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China)

Correspondence to:WANG Xin,Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China (Tel: 86-531-85186358. Fax:86-531-87068707. E-mail:xinwang55@yahoo.com.cn)
Keywords
polycythaemia vera; regulatory T cells; pathogenesis
Abstract

Background  Regulatory T cells (Treg) have been shown to play an important role in the regulation of hematopoietic activity. However, there is no information about the effect of Treg cells in the pathogenesis of polycythaemia vera (PV).
Methods  In this study, we investigated the percentage and function of Treg cells in the peripheral blood of 21 PV patients and 25 healthy donors. Treg cells were identified and characterized as CD4+CD25+FOXP3+ by flow cytometry. The suppressive activity of CD4+CD25+ Treg cells was assessed by the proliferation and cytokine secretion of the co-cultured CD4+CD25 fractions.
Results  The results showed that the percentage of Treg cells in the peripheral blood of PV patients significantly increased compared to healthy controls ((10.93±4.02)% vs (5.86±1.99)%, P <0.05). Moreover, the mRNA and protein expression of FOXP3 was higher in CD4+CD25+ Treg cells. Coordinately, when co-cultured with the activated CD4+CD25 cells, the CD4+CD25+ Treg cells showed enhanced suppressive function in PV. Yet, the underlying mechanism for the increased frequency and function of CD4+CD25+ Treg cells is still to be clarified.
Conclusion  Treg cells expansion might account for the abnormal T cell immunity in PV patients and thus contribute to the pathogenesis of PV.

Polycythaemia vera (PV) is one of the chronic myeloproliferative diseases arising from the clonal expansion of a pluripotent hematopoietic progenitor cell that causes the accumulation of morphologically normal red cells, white cells, platelets, and their progenitors in the absence of a definable stimulus and to the exclusion of non-clonal hematopoiesis. It is not a rare disease with an estimated incidence of about 2.3 per 100 000 every year.1 The median age of presentation is approximately 60 years old.2 PV is associated with significant morbidity and mortality. Without treatments, it can lead to thrombo-hemorrhagic complications and in most cases to progressive myelofibrosis, anemia, and acute myeloid leukemia.

The etiology of PV remains unknown and there is no consensus as to the optimal therapy for the disorder. Aggressive phlebotomy therapy remains the mainstay of initial treatment of PV but unfortunately had a high incidence of early thromboses.3 Neither chemotherapy nor 32P was observed superior to phlebotomy in preventing thrombosis but both have proved to be more toxic.4 Recently, a novel somatic single point mutation in the 9p chromosomal region encoding the tyrosine kinase JAK2 has been reported in several myeloproliferative diseases (MPDs). The JAK2V617F allele has also been identified in the hematopoietic stem cell (HSC) compartment in patients with PV.5 This recurrent somatic mutation results in constitutive activation of the JAK2 tyrosine kinase and leads to cytokine hypersensitivity and erythrocytosis.6,7 Nevertheless, the JAK2V7617F was not detected in B or T lymphocyte fractions, allowing the possibility that the target population was not multipotent but myeloid-committed.8,9 However, there was also report that the JAK2V7617F mutation arises not as a PV-initiating mutation.10 Further investigations are needed for the understanding of the pathogenesis of the disease.

Regulatory T cells (Treg cells) play a vital role in the maintenance of self-tolerance, control of auto-immunity and regulation of T-cell homeostasis, and they modulate overall immune responses against a variety of pathogens. Natural T regulatory cells are a subgroup of CD4+ T cells that constitutively express IL-2 receptor α chains (CD25) in the resting state. Treg cells are characterized for their anergic state. They also possess ability to actively inhibit CD4+CD25 T cells, CD8+ T cells, dendritic cells (DC), natural killer cells (NK), natural killer T cells (NKT), and B cells in a cell-cell contact and dose-dependent manner.11-14 The transcription factor forkhead box P3 (FOXP3) is specially expressed in CD4+CD25+ natural Treg cells and is required for the function of Treg cells.15 In the past few years, the involvement of naturally arising Treg cell subsets in blood diseases have been studied and there is accumulating evidence that these cells play a part in the process of hematopoietic activity.

Currently, no information on the role of Treg cells and the mechanism in PV is available. However, a significant impairment of T cell function was observed in patients with PV.16 Considering Treg cells might participate in the dysfunction of T cell immunity in PV, we evaluated the profile and function of Treg cell subsets in the blood of patients with PV to seek for the possible relation between Treg cell immunity and pathogenesis of PV. We also pursued to explore the possibility for the treatment clue for PV.

METHODS

Patients
With the approval of the Health Service Ethics Committee of Shandong Provincial Hospital, peripheral blood samples for test group were collected from 21 patients with PV (13 male, 8 female) with an average age of (55±3) years old (rang, 14–72 years) and 25 healthy individuals (16 male, 9 female) with an average age of (50±2) years old (range, 21–67 years) were taken as control. Samples were obtained after informed consent. Diagnoses of PV were made according to the WHO criteria.17 All the subjects were negative for antinuclear antibody and HIV serological tests. The peripheral blood samples of test group were collected from PV patients prior to the commencement of any treatment.

Mononuclear cells isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole peripheral blood by Ficoll/Hypaque (Chinese Academy of Medical Sciences, Beijing, China) density centrifugation at 400×g for 20 minutes for immediate use or stored in liquid nitrogen until further use. At least 2×106 cells were prepared for flow cytometry analysis.

Flow cytometry analysis
Three-color flow cytometry analyses were conducted in PBMCs isolated from healthy donors and patients with PV for CD4+CD25+FOXP3+ Treg cell frequencies. About 100 µl prepared cells (1×106) were stained with anti-CD4-PC5 and anti-CD25-FITC monoclonal antibodies. Then a Fixation/Permeability working solution was added before the intracellular staining of anti-human FOXP3-PE. IgG1-FITC and IgG2a-PE were used as isotype controls. Flow cytometry was performed on a Becton Dickinson FACSCalibur with the CellQuest software. The antibodies and fixation/permeability working solution were purchased from eBiosciences, USA.

Cell sorting
For functional analysis, CD4+CD25+ T cells were sorted from fresh PBMCs using a CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacture’s instructions. In brief, non-CD4+ cells were labeled with a cocktail of biotin-conjugated antibodies and anti-biotin magnetic beads. The labeled cells were subsequently depleted by separation over a MACS column. Then purified CD4+ T cells were incubated with anti-human CD25 magnetic beads and isolated by positive selection from the pre-enriched CD4+ T cell fraction. The magnetically labeled CD4+CD25+ T cells were retained on the column and eluted after removal of the column from the magnetic field. The sorted CD4+CD25+ cells were consistently with over 90% FOXP3+ by flow cytometry analysis. The purified CD4+CD25 cells were used for later experiments.

Proliferation and suppressive activity of CD4+CD25+ Treg cells
CD4+CD25 and CD4+CD25+ cells were sorted and purified as described above. To determine the suppressive ability of CD4+CD25+ T cells, CD4+CD25 cells (105/well) were cultured and stimulated with plate-bound anti-CD3 (eBiosciences) and 5 mg/L soluble anti-CD28 (BD PharMingen, USA) in the presence of 1×104 units/L interleukin (IL)-2 (R&D Systems, USA) in 96-well round-bottomed plates for 72 hours. CD4+CD25 cells were co-cultured with CD4+CD25+ cells at the ratio of 1:0, 4:1, 2:1, 1:1, and 0:1. Proliferation rate was measured after 72 hours by [3H]-incorporation. Then 0.5 µCi/well [3H]-thymidine (Amersham Pharmacia, Little Chalfont, United Kingdom) was added to the cultures and cell proliferation was measured by incorporation of radio labeled thymidine for 16 to 18 hours. Incorporated radioactivity per minute was measured using a scintillation counter (Wallac, Turku, Finland). Culture supernatants from the proliferation assays were removed before addition of [3H]-thymidine and tested for interferon (IFN)-γ with an enzyme-linked immunosorbent assay (ELISA) kit (Biolegend, USA) according to the manufacture’s instructions.

Real-time PCR
Total RNA was isolated from the CD4+CD25+ and CD4+CD25 cells using Trizol reagent (Invitrogen, USA) and the ratio of A260/280 was controlled always >1.6. The integrity of RNA was confirmed by the presence of intact 18S and 28S bands on 1% agarose gel. The first-strand cDNA synthesis reaction was performed using Fermantas Reverse Transcription kit (MBI Fermentas, Vilnius, Lithuania) according to the manufacture’s instructions. The mRNA and cDNA were stored at –80˚C until use. Applied Biosystems SYBR Green Master Mix Kit (Tiangen Biotech, Beijing, China) was used to examine the expression of the mRNA of FOXP3 level using the primers according to the references:18 FOXP3 sense 5′-TCTGTGGCATCATCCGACAA-3′ and antisense 5′-AACTCTGGGAATGTGCTGTTTC-3′; taking β-actin as an inner control (sense 5′-AATGCTTCTAGGCGGAC- TATGA-3′ and antisense 5′-CAAGAAAGGGTGTAA- CGCAACT-3′) under the following conditions: 1 cycle at 95˚C for 5 minutes, then 30 cycles at 94˚C for 30 seconds and 60˚C for 45 seconds, then 72˚C for 30 seconds and 4˚C withhold. The mRNA in each sample was automatically quantitied with reference to the standard curve conducted each time using ABI 7000 software. Quantitative RT-PCR was performed at least 3 times, including a no-template sample as a negative control.

Western blotting
Proteins from CD4+CD25+ cells were extracted using a protein extraction kit (Biocolor Biotech, Shanghai, China) and electrophoresed on 10% SDS polyacrylamide gels and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was incubated overnight with the mouse anti-human FOXP3 antibody (diluted 1:1000, eBiosciences) and subsequently incubated with an anti-mouse secondary antibody (diluted 1:50, Dako, Glostrup, Denmark) for 1 hour at room temperature. Proteins were re-blotted using anti-human GAPDH (diluted 1:10000, Zymed Lab, San Francisco, CA). Protein bands were visualized by SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, USA).

Statistical analysis
Data were expressed as means ± standard error (SE). Statistical analysis was done by Mann-Whitney U test to assess differences between the PV and healthy control groups. Correlations among the parameters were analyzed by the Whitney U test. P <0.05 was considered statistically significant. All statistical analyses were performed using the SPSS statistical software package (SPSS 11.5 for windows; SPSS, Chicago, IL, USA).

RESULTS

Clinical characteristics of PV patients and healthy controls
We analyzed 21 cases of PV patients and 25 healthy donors for the prevalence of Treg cells. The clinical characteristics of the two groups were summarized in Table 1. There was no statistical significance between the two groups in the aspects of cases, age, or sex. But for the blood parameters, the PV group had significantly higher blood cells, haematocrit (Hct) and neutrophil alkaline phosphatases (NAP) counts than the healthy controls (P <0.05).
 

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Table 1. Clinical parameters and Treg percentages of PV patients and healthy control

PV patients have higher percentage of Treg cells in the peripheral blood
The population of CD4+CD25+FOXP3+ T cells, as a representative example for the definition of cell proportion, was shown in Figure 1. The second quadrant represents the positive percentage of staining. The percentage of CD4+ T cells was significantly lower than in normal control ((28.7±7.1)% vs (38.6±8.4)%, P <0.05); however, the percentage of CD4+CD25+FOXP3+ T cells in PV was significantly higher than healthy donors ((10.9±4.0)% vs (5.9±2.0)%, P <0.001). Statistical analysis did not show significant correlation of age, sex with percentage of Treg cells, while the Treg cells level was correlated with CD4+ T cell percentage (n=21, r=0.61, P=0.003).
 

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Figure 1. Increase of CD4+CD25+ lymphocytes in peripheral blood of PV patients. Representative examples in flow cytometry analysis are of cells from PV patients (AC) and healthy donors (DF). Cells were stained with anti-CD4-PC5, anti- CD25-PE and anti-FOXP3-FITC antibodies. A and D: frequency of CD25+FOXP3+ cells in CD4+ T cells; B and E: frequency of CD25+ cells in the CD4+ T cells; C and F: frequency of FOXP3+ cells in CD4+ T cells. PV: polycythaemia vera.

FOXP3 mRNA expression in CD4+CD25+ Treg cells
To investigate the mRNA expression of FOXP3 in CD4+CD25+ and CD4+CD25 T cells, real-time PCR was performed with mRNA extracted from purified CD4+CD25+ and CD4+CD25 cells sorted from 15 representative PV patients and 10 healthy controls. The relative mRNA expression level of PV group is about 3-fold more than that of control group in CD4+CD25+ T cells, whereas FOXP3 mRNA expression can hardly detected in CD4+CD25 T cells. The average FOXP3 mRNA expression level in PV was significantly higher than in healthy donors (Figure 2).


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Figure 2. FOXP3 mRNA expression analyzed by real-time PCR. The expression of FOXP3 in CD4+CD25 and CD4+CD25+ cells from PV patients and healthy donors using real-time PCR. The relative FOXP3 mRNA expression of sorted CD4+CD25+ cells from both PV patients and healthy donors is respectively 64.23±18.52 and 16.06±4.78 (*P <0.05). FOXP3 mRNA expression was significantly lower in CD+CD25- cells compared with CD4+CD25+ cells in controls (**P <0.05). PV: polycythaemia vera.

Expression of FOXP3 in Treg cells of PV at protein level
Western blot analysis of FOXP3 expression in CD4+CD25+ Treg cells from 5 PV patients and 5 healthy donors tended to parallel the results of relative mRNA expression with quantitative real time PCR. FOXP3 was specifically expressed in CD4+CD25+ Treg cells (Figure 3), the FOXP3 is higher in PV patients when compared to healthy control (P <0.05). These results also further confirmed the cells we sorted before were really Treg cells.


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Figure 3. FOXP3 protein expression in CD4+CD25+ cells by Western blotting analysis. The protein level of FOXP3 in CD4+CD25+ cells is higher in PV patients compared to healthy controls (P <0.05). All experiments were repeated for three times, and typical data from one of three experiments are shown.

Suppressive activity of CD4+CD25+ Treg cells from PV patients
To investigate whether the increased Treg cells were associated with a functional effect, the suppression activity of Treg cells on mixed lymphocytes reaction were performed in which CD4+CD25 T cells were cultured in the absence or presence of Treg cells at the ratio of 1:0, 4:1, 2:1, 1:1 and 0:1. We observed a strong inhibition of the T-cell proliferation by CD4+CD25+ T cells from PV patients (Table 2). The inhibition rate was highest at the ratio of 1:1. However, the CD4+CD25+ T cells of PV patients have stronger inhibition than healthy controls under low concentrations such as 4:1 and 2:1. At a ratio of 1:1, CD4+CD25+ suppressed the proliferation of responder cells by an average rate of (20.5±3.0)% vs (25.5±4.5)% in PV patients and healthy control (P <0.05).


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Table 2. Suppressive function of CD4+CD25+ T cells from peripheral blood of PV and healthy control (%)

Cytokine expression of CD4+CD25 T Cells
Freshly isolated CD4+CD25 and CD4+CD25+ cells from PV patients and control groups were stimulated with plate-bound anti-CD3 and soluble anti-CD28 and IL-2 for 72 hours as described above. The culture supernatants were assayed for IFN-γ (Table 3). The data demonstrate that concentration of IFN-γ is correlated with the suppressive level of Treg cells from PV patients. CD4+CD25 T cells from PV patients predominantly secrete IFN-γ whereas CD4+CD25+ T cells mainly produce very low levels of IFN-γ. It was shown that the IFN-γ production decreased whenever the proliferation was inhibited. At the ratio of 1:1, concentration of IFN-γ was (65.9±11.4)% versus (91.2±6.5)% in PV and healthy control (P <0.05).
 

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Table 3. IFN-γ content in supernatant of co-cultured CD4+CD25/ CD4+CD25+ cells (ng/L)

DISCUSSION

In this study, we investigated the percentage and the function of Treg cells in peripheral blood of 21 PV patients and 25 healthy controls. Interestingly, we found that CD4+CD25+ Treg cells in PV patients were significantly increased in percentage and function compared to healthy controls.

We compared the frequency of CD4+CD25+FOXP3+ Treg cells in PV and healthy controls. The CD4+ T cells were decreased while CD4+CD25+FOXP3+ Treg cells were significantly increased within the CD4+ T cells in PV when compared to the controls. Classically, the phenotypic characters of Treg cells were CD4, CD25 and FOXP3.19,20 In that view, we used the molecule marker of CD4, CD25 and FOXP3 by flow cytometry analysis to identify Treg cells more precisely. This allows for the exclusion of other activated CD4+ T cells in peripheral blood.

The decrease of CD4+ T cells in PV suggested deficiency in T cell immunity and a possible inhibitory function that might relate to the increased CD4+CD25+ Treg cells in PV patients. It is increasingly accepted that immune system plays an important role in the regulation of hematopoiesis. T cells can produce a diverse set of cytokines and regulate the function of committed hematopoietic under conditions of immune activation.21 However, decreased T cell responses to mitogenic and T-cell receptor-mediated stimulations and an altered CD4/CD8 ratio due to the decreased CD8 subpopulation were observed in PV patients.16,22 The impairment of T cell immunity in PV suggested a possible physiological inhibitory mechanism as suppressor T cells such as Treg cells and some cytokines can potentially inhibit auto-reactive T and B cells from activated.

Previous studies showed a percentage of 5% to 10% of Treg cells to CD4+ T cells in healthy control.23 Yet, the staining of Treg cells in those studies is CD4+CD25+ or CD25high, which could not precisely reflect CD4+CD25+ Treg cells. FOXP3 is specifically expressed in CD4+CD25+ Treg cells and is required for regulatory and suppressive function. Moreover, although FOXP3 is also found in CD4+CD25 subsets, it was indicated that these suppressive FOXP3+CD4+CD25 T cells were either very small or much potent. Anyway, the detection of FOXP3 in CD4+CD25+ T cells is more reliable to reflect the profile of the CD4+CD25+ Treg cells. FOXP3 was hardly expressed in CD4+CD25 cells but in CD4+CD25+ T cells from PV patients was up-regulated at both mRNA and protein level. These results confirmed that the CD4+CD25+ T cells isolated in our study were really Treg cells. The up-regulation of FOXP3 and Treg cell frequency implicated a stronger inhibition effect of the increased CD4+CD25+ Treg cells.

In the in vitro assays, we also studied the function of CD4+CD25+ Treg cells in PV patients by analyzing the proliferative and suppressive activities as well as cytokine profile. The CD4+CD25+ T cells from PV patients inhibited the proliferative response of CD4+CD25 T cells significantly upon T cell receptor stimulation. And even at low levels CD4+CD25+ Treg cells have stronger suppression effect compared to healthy control. The suppressive effect of CD4+CD25+ Treg cells was further confirmed with a reduced level of IFN-γ production by CD4+CD25 cells co-cultured with CD4+CD25+ T cells in vitro. Thus, the Treg cells in PV had a strengthened suppressive effect and this was coincided with the up-regulation of FOXP3 expression. It was suggested that CD4+CD25+ Treg cell suppression is cell contact dependent or mediated by cytokines. Treg cells are able to suppress the secretion of IL-2 by CD4+CD25 T cells, thus weakening activation signals for cytotoxic cells.24 Moreover, Treg cells can down-regulate the cytotoxic activity of CD8+ and NK cells with direct contact with these cells.11,12

Yet, the biologic basis for the increased number and function of CD4+CD25+ Treg cells in PV remains undefined. Recent studies have reported an increase in number of Treg cells in patients with a variety of blood diseases.25-27 It was suggested that the CD4+ Treg cell expansion is a feature of high risk MDS and progression to aggressive subtypes of the disease. Update study suggested that CD4+CD25+ Treg cells could alter hematopoietic progenitor cell activity in a direct cell-to-cell way or via production of effector cytokines.28 Treg cells have been shown to suppress T cell immunity and down-regulate the natural killer cell immunity in vivo by a bone marrow transplantation model.29,30 Therefore, our preliminary data present evidence that the elevated Treg cells in PV might be activated and then affect the hematopoietic activity. We believe that Treg cells might account for the dysfunction of T lymphocytes and NK cells in their disability to down-regulate the hematopoietic proliferation in PV. However, further experimental models should be constructed to study whether the elimination of CD4+CD25+ T cells lead to effective anti-proliferative immune responses.

In conclusion, we found profound abnormalities of Treg cells in patients with PV and these findings confirmed and extended earlier studies. The most important finding was an increase in the percentage of CD4+CD25+FOXP3+ Treg cells in the peripheral blood of PV patients. This was substantiated with the up-regulation of FOXP3 within CD4+CD25+ T cells in PV both at mRNA and protein level. We recommended that natural Treg cells play an important role in the PV, but the biological basis and further mechanism of the Treg cells in patients with PV remain undefined. Further investigation of this abnormality might provide novel therapy clues for this disease.

Acknowledgments: We thank the medical and nursing staff in the Department of Hematology at Shandong Provincial Hospital for providing clinical samples. We thank Dr. WU Nan for her helpful consultations for the trials and manuscript. We also thank Prof. YOU Li, MA Chun-yan and CUI Bin for their technical assistance.

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