Increased frequencies of T helper type 17 cells in the peripheral blood of patients with acute myeloid leukaemia

Authors


H. Xu, Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, 212013 Zhenjiang, China.
E-mail: xuhx@ujs.edu.cn

J. Jin, Department of Cell Biology, Medical College, Jiangsu University, 212013 Zhenjiang, China.
E-mail: Jinjie9@163.com

Summary

T helper type 17 (Th17) cells, which represent a novel subset of CD4+ T cells, play an active role in inflammatory and autoimmune diseases. Recent studies have also suggested that they have an impact on solid tumours. However, the nature of Th17 cells in haematological malignancies remains unknown. In this study, we investigated Th17 cell frequency and secretion of related cytokines in patients with acute myeloid leukaemia (AML). First, we found that Th17 cell frequencies were increased significantly in peripheral blood samples from untreated patients with AML, compared with those from healthy volunteers. Moreover, increased interleukin (IL)-17 concentrations accompanied the increased Th17 cell frequencies in these patients. These results suggest that Th17 cells may play a role in the pathogenesis of AML. Secondly, we found that the increased Th17 cell frequencies were reduced when patients achieved complete remission after chemotherapy, suggesting that measurement of Th17 cell frequencies may have clinical value in the evaluation of therapeutic effect. In addition, we found that IL-6 and transforming growth factor (TGF)-β1 concentrations increased in the untreated patients and that IL-6 concentrations showed a positive correlation with the frequencies of Th17 cells, suggesting that IL-6 may play an important role in Th17 cell differentiation in patients with AML.

Introduction

Acute myeloid leukaemia (AML) is characterized by the proliferation of clonal neoplastic myeloid haematopoietic precursor cells and impaired production of normal haematopoiesis. It accounts for 80% of acute leukaemia in adults [1]. Although the involvement of immune system impairment in the pathogenesis of AML has long been recognized, the explicit mechanism remains unknown.

CD4+T cells are essential regulators of the anti-tumour immune response, directing the differentiation of other immune cells in response to a variety of tumour antigens. Based on cell function, CD4+ T cells are divided traditionally into three major subsets: T helper type 1 (Th1), Th2 and regulatory T (Treg) cells [2]. More recently, a unique CD4+ T cell subset named Th17 has been described [3]. Th17 cells have been shown to play a crucial role in the development of inflammatory diseases, autoimmune diseases and graft-versus-host disease (GVHD) by producing interleukin (IL)-17A and other cytokines, including another member of the IL-17 family known as IL-17F [4–6]. In mice, the differentiation of Th17 cells are driven primarily by the cytokines transforming growth factor (TGF)-β and IL-6, whereas IL-23, thought originally to be the master regulator, seems to be important for maintenance of Th17 responses [7–9]. Retinoic acid-related orphan nuclear receptor gamma t (ROR-γt) is a transcription factor that is considered to be important for the initiation and maintenance of the Th17 cell lineage [10,11]. However, less is known in humans than in mice. Volpe et al.[12] have reported that TGF-β, IL-23 and proinflammatory cytokines (IL-1β and IL-6) were all essential for human Th17 differentiation. Nevertheless, Acosta-Rodriguez et al.[13] have found that for human naive CD4+ T cells, ROR-γt expression and Th17 polarization were induced by IL-1β and enhanced by IL-6 but were suppressed by TGF-β and IL-12.

Accumulating evidence indicates that an imbalance in Th1/Th2 cells and an increase of Treg cells are involved in the pathogenesis of solid tumours [14–16] as well as haematological malignancies [17,18], but the role of Th17 cells in tumour immunity has been poorly defined. A few reports indicate that the number of Th17 cells in patients with solid tumours was increased [19,20]. However, the nature and regulation of Th17 cells in patients with AML have not been reported.

In this study, we evaluated the frequencies of Th17 cells and the concentrations of related cytokines (IL-17, IL-6 and TGF-β1) in patients with AML, allowing for analysis of their involvement in the pathogenesis of AML.

Patients and methods

Patients

Forty-two patients diagnosed newly with AML treated at the Affiliated People's Hospital of Jiangsu University were included in this study from 2007 to 2009. Of these patients, 27 achieved complete remission (CR) after one or two courses of standard induction chemotherapy [21], with the remaining patients failing to achieve CR after two courses of standard induction chemotherapy. Diagnoses were established according to the French–American–British (FAB) classification system [22,23]. CR was defined based on International Working Group criteria [24]. Thirty-six healthy volunteers were studied simultaneously as controls. Data describing the study subjects are summarized in Table 1. This study was approved by the ethical committee of the Affiliated People's Hospital of Jiangsu University, and written informed consent was obtained from all individuals.

Table 1.  Subjects' characteristics.
 Untreated patients
(n = 42)
CR patients
(n = 27)
Controls
(n = 36)
  1. CR, complete remission; FAB, French–American–British; s.d., standard deviation.

Age (years, mean ± s.d.)48·12 ± 3·3043·37 ± 4·0646·38 ± 3·28
Gender (male/female)23/1915/1219/17
FAB subtype   
 M132 
 M2159 
 M396 
 M4128 
 M532 

Blood samples

Peripheral blood samples were collected from healthy volunteers and patients before and after one or two courses of induction chemotherapy. The collection tubes contained 0·2 ml sodium heparin. Peripheral blood mononuclear cells (PBMCs) were obtained by standard Ficoll-Hypaque density centrifugation for analysis by flow cytometry. Plasma was obtained after centrifugation and stored at −80°C for measurement of cytokine levels.

Flow cytometric analysis of Th17 cells

PBMCs (2 × 106 cells/ml) were suspended in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine (Invitrogen, Carlsbad, CA, USA). The cells were seeded into 24-well plates and stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml ionomycin for 4 h in the presence of 500 ng/ml monensin (Alexis Biochemicals, San Diego, CA, USA). After 4 h of culture at 37°C under 5% CO2, the cells were transferred to sterile tubes and centrifuged at 800 g for 5 min. After being washed once in phosphate-buffered saline (PBS), the cells were incubated at 4°C for 20 min for surface staining with the following anti-human monoclonal antibodies (BD PharMingen, San Diego, CA, USA): anti-CD3-allophycocyanin (APC) and anti-CD8-peridinin chlorophyll protein (PerCP). The cells were then stained with anti-IL-17-fluorescein isothiocyanate (FITC) (BD PharMingen) for Th17 detection, after fixation and permeabilization according to the manufacturer's protocol. Stained cells were analysed by flow cytometric analysis using a fluorescence activated cell sorter (FACS)Calibur cytometer (BD Pharmingen) equipped with CellQuest software.

Enzyme-linked immunosorbent assays (ELISAs) for plasma IL-17, IL-6 and TGF-β

Plasma levels of IL-17, IL-6 and TGF-β1 were measured by ELISAs, following the manufacturer's protocols (eBioscience, San Diego, CA, USA). All samples were measured in triplicate.

Statistical analysis

Values are expressed as means ± standard deviation (s.d.) in the text and figures. Comparisons between paired or unpaired groups were performed using the appropriate Student's t-test. Spearman's correlation was used to test correlation between two continuous variables. P < 0·05 was considered to be statistically significant.

Results

Increased frequencies of Th17 cells in PBMCs from untreated patients with AML

Flow cytometry was used to assess frequencies of Th17 cells in PBMCs from untreated AML patients and controls (Fig. 1). Because stimulating PBMCs with PMA down-regulates the surface expression of CD4 by internalization [25], according to several previous studies [26,27] and the results of preliminary experiments, CD3+CD8 T cells were considered CD4+ T cells, as shown in Fig. 1(a1). Preliminary experiments have shown that these two populations are >96% congruent in AML patients and controls (data not shown). Representative plots showed that the population of Th17 cells in CD3+CD8T cells increased in an untreated patient compared with that in a healthy volunteer, as shown in Fig. 1(a2,a3). Figure 1b shows that the frequencies of Th17 cells (CD3+CD8IL-17+/CD3+CD8T cells) in untreated patients (3·22 ± 0·26%) were higher than those in controls (0·88 ± 0·16%), where the difference was statistically significant (P < 0·01). Our data indicated that the expansion of Th17 cells described previously in patients with solid tumours [19,20] was also apparent in AML patients.

Figure 1.

Increased frequencies of T helper type 17 (Th17) cells (CD3+CD8IL-17+/CD3+CD8T cells) in peripheral blood from untreated patients with acute myeloid leukaemia (AML). Peripheral blood mononuclear cells (PBMCs) from untreated AML patients (n = 42) and healthy volunteers (n = 36) were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 h in the presence of monensin and then stained with labelled antibodies as described in Methods. The stained cells were measured by flow cytometry. (a) Representative flow cytometry plots of Th17 cells. (a1) CD3+CD8 T cells were considered CD4+ T cells in region R4. (a2) Representative interleukin (IL)-17 expression in CD3+CD8 T subsets from an untreated patient with AML. (a3) Representative IL-17 expression in CD3+CD8 T subsets from a healthy volunteer). (b) Collective analysis of results from two groups. Results were expressed as mean ± standard deviation. *P < 0·01.

Increased cytokine concentrations in plasma from untreated patients with AML

Concentrations of plasma IL-17, IL-6 and TGF-β1 measured by ELISAs in each group are shown in Table 2. IL -17, IL-6 and TGF-β1 concentrations in untreated patients with AML were higher than those in controls, respectively. These differences were statistically significant (P < 0·05, P < 0·01 and P < 0·01, respectively). Additionally, the IL-6 concentrations showed a positive correlation with the frequencies of Th17 cells (r = 0·54, P < 0·01), whereas the TGF-β1 concentrations did not show any correlation with the frequencies of Th17 cells in untreated patients (r = 0·23, P > 0·05), as shown in Fig. 2.

Table 2.  Plasma levels of cytokines in untreated patients and controls (means ± standard deviation).
 Untreated patients
(n = 42)
Controls
(n = 36)
P
  1. IL: interleukin; TGF: transforming growth factor.

IL-17 (pg/ml)18·65 ± 3·1910·52 ± 1·69<0·05
IL-6 (pg/ml)15·22 ± 3·861·44 ± 0·48<0·01
TGF-β1 (ng/ml)51·37 ± 11·3014·35 ± 4·00<0·01
Figure 2.

Spearman's correlation of T helper type 17 (Th17) cell frequencies and related cytokine concentrations in untreated patients with acute myeloid leukaemia (AML) (n = 42). (a) Interleukin (IL)-6 concentrations correlated positively with Th17 cell frequencies (r = 0·54, P < 0·01). (b) Transforming growth factor (TGF)-β1 concentrations did not correlate with Th17 cell frequencies (r = 0·23, P > 0·05).

Reduced frequencies of Th17 cells in PBMCs from AML patients achieving CR after chemotherapy

After treatment with chemotherapy, 27 patients achieved CR and 15 patients failed to achieve CR. Frequencies of Th17 cells in PBMCs from the CR patients and non-CR patients were measured by flow cytometry. As shown in Fig. 3, the frequencies of Th17 cells were reduced significantly in CR patients (1·23 ± 0·21%) compared with the same patients before treatment (2·98 ± 0·35%). The difference was statistically significant (P < 0·01). However, in non-CR patients, frequencies of Th17 cells (2·54 ± 0·51%) were not reduced significantly compared with those in the patients before treatment (3·64 ± 0·37%). The difference was not statistically significant (P > 0·05).

Figure 3.

Comparison of T helper type 17 (Th17) cell frequencies in peripheral blood from patients before and after treatment with chemotherapy. Peripheral blood mononuclear cells (PBMCs) from complete remission (CR) patients (n = 27) and non-CR patients (n = 15) with acute myeloid leukaemia (AML) were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 h in the presence of monensin and then stained with labelled antibodies as described in Methods. The stained cells were measured by flow cytometry. Frequencies of Th17 cells were reduced significantly in CR patients compared to frequencies in those patients before treatment. Frequencies of Th17 cells were not reduced in non-CR patients compared to frequencies in those patients before treatment. *P < 0·01.

Discussion

Th17 cells and their effector cytokines are being recognized increasingly as important mediators in inflammatory and autoimmune diseases, but relatively little is known about their specific roles in human tumour immunity. Kryczek et al. have reported increased numbers of Th17 cells in peripheral blood in patients with advanced ovarian carcinoma and in tumour tissues in patients with advanced ovarian, pancreatic and renal cell carcinomas [19]. Zhang et al. have also found that patients with gastric cancer had a higher proportion of Th17 cells in peripheral blood and that the increased prevalence of Th17 cells was associated with clinical stage [20]. These findings suggest that Th17 cells may play a role in the pathogenesis of solid tumours. Nevertheless, Wrobel et al. have demonstrated that serum levels of IL-17 are not elevated in AML patients [28]. To assess whether Th17 cells contribute to the pathogenesis of AML, we studied different characteristics of Th17 cells in AML patients, including cell frequency and related cytokine secretion. The results were consistent with studies from patients with solid tumours [19,20], but not consistent with results from the study by Wrobel et al.[28]. In the study by Wrobel et al., only 10 samples from healthy volunteers as controls may be not adequate to show a difference between AML patients and controls. Some studies in animals have demonstrated that IL-17 may promote angiogenesis and tumour growth [29,30]. Hence, the increased number of Th17 cells in AML patients may promote the development or progress of AML through secretion of IL-17. On the other hand, Muranski et al. found more recently that tumour-specific Th17-polarized cells could eradicate large, established melanomas in mice [31]. That is, Th17 cells exerted an anti-tumour effect. In light of these results, the increase in Th17 cells in AML patients may be explained as a protective reaction of the immune system in certain stages of the disease. In any case, Th17 cells may participate in the development or progress of AML. In addition, in the current study, we have also found that the increased Th17 cell frequencies in untreated patients were reduced when those patients achieved CR after chemotherapy, suggesting that measurement of Th17 cell frequency may be valuable as an evaluation of therapeutic effect. These results also suggest that the number of Th17 cells may relate to tumour burden, which is an important prognostic factor in a variety of tumour types [32]. Whether the measurement of Th17 cell numbers has prognostic significance for AML needs further research. In this study, our data provide some valuable information to support further research on the precise involvement of Th17 cells in AML.

Most of the knowledge of Th17 cell differentiation available to date originates from experimental animals, whereas very little information exists about human Th17 cells. In mice, TGF-β is the cytokine most critical for Th17 initiation, and IL-6 acts as a critical co-factor for Th17 cell differentiation [7–9]. In humans, different studies have revealed that the role of TGF-β may not be central to Th17 differentiation, but IL-1, IL-23 or IL-6 may have an important role [13,33,34]. However, very recently, three groups reported independently that TGF-β was also critical for human Th17 cell differentiation [12,35,36]. They have shown a requirement for low doses of TGF-β together with various combinations of IL-1β, IL-6, IL-21 and IL-23 in human Th17 cell differentiation. In our study, results showed that the increased concentrations of IL-6 correlated positively with the frequencies of Th17 cells in those patients, but changes in TGF-β1 concentration did not show a correlation with the frequencies of Th17 cells. These results suggest that, in the presence of TGF-β1 at certain levels, Th17 cell numbers may increase along with the elevation of IL-6 concentration in AML patients. Therefore, our data indicate that IL-6 plays an important role in Th17 cell differentiation in patients with AML, whereas the role of TGF-β needs to be clarified further.

In brief, our data provide a strong association between increased Th17 activity and AML, which opens a new avenue in the study of tumour immunotherapy. However, the precise involvement of Th17 cells in tumour pathogenesis needs clarification. Future research should address this interesting issue further.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (30871193 and 30671984), the Society Development Foundation of Zhenjiang (SH2006031), Top Talent Project of Jiangsu University and SCI-Tech Innovation Team of Jiangsu University (2008-018-02).

Disclosure

None.

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