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IL-17/IL-10 double-producing T cells: new link between infections, immunosuppression and acute myeloid leukemia

  • 1,
  • 2,
  • 2,
  • 3,
  • 3,
  • 2,
  • 1,
  • 1,
  • 1,
  • 1,
  • 1,
  • 3,
  • 4,
  • 5,
  • 1,
  • 6,
  • 3,
  • 2,
  • 7 and
  • 2Email author
Contributed equally
Journal of Translational Medicine201513:229

  • Received: 6 May 2015
  • Accepted: 29 June 2015
  • Published:



Acute myeloid leukemia (AML) is an incurable disease with fatal infections or relapse being the main causes of death in most cases. In particular, the severe infections occurring in these patients before or during any treatment suggest an intrinsic alteration of the immune system. In this respect, IL-17-producing T helper (Th17) besides playing a key role in regulating inflammatory response, tumor growth and autoimmune diseases, have been shown to protect against bacterial and fungal pathogens. However, the role of Th17 cells in AML has not yet been clarified.


T cell frequencies were assessed by flow cytometry in the peripheral blood of 30 newly diagnosed AML patients and 30 age-matched healthy volunteers. Cytokine production was determined before and after culture of T cells with either Candida Albicans or AML blasts. Statistical analyses were carried out using the paired and unpaired two-tailed Student’s t tests and confirmed with the non parametric Wilcoxon signed-rank test.


A strong increase of Th17 cells producing immunosuppressive IL-10 was observed in AML patients compared with healthy donors. In addition, stimulation of AML-derived T cells with a Candida albicans antigen induced significantly lower IFN-γ production than that observed in healthy donors; intriguingly, depletion of patient Th17 cells restored IFN-γ production after stimulation. To address the role of AML blasts in inducing Th17 alterations, CD4+ cells from healthy donors were co-cultured with CD33+ blasts: data obtained showed that AML blasts induce in healthy donors levels of IL-10-producing Th17 cells similar to those observed in patients.


In AML patients altered Th17 cells actively cause an immunosuppressive state that may promote infections and probably tumor escape. Th17 cells could thus represent a new target to improve AML immunotherapy.


  • AML
  • Infections
  • IL17+/IL10+ T cells
  • Immunosuppression
  • Leukemia immunoescape


Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults [1] and current treatments remain unsatisfactory. Serious infections, resistance to therapy and relapses are the main causes of mortality among patients [24]. In particular, the high frequency and severity of infections (especially fungal) before or during chemotherapy are probably due to a severe adaptive immunity dysfunction directly induced by the disease [5, 6]. This immunosuppressive state may also be responsible for the continuous recurrence of AML and for the failure of immunotherapies [7], with the exception of allogeneic transplantation [8].

Although several studies identified different immunosuppressive mechanisms operating in AML [913], the precise link between immune alterations, leukemia immune escape and infections has not yet been elucidated. Investigating in this direction, T helper cells seem to be important players with Th17 cells being one of the most intriguing and not fully understood subset so far [14].

Th17 cells show a pleiotropic role in the inflammatory response, autoimmune disorders and tumors [1519]. In the latter context, they showed remarkable epigenetic plasticity [14] and the ability to transdifferentiate into T helper 1-like cells (secreting IFN-γ and showing tumor suppressor activity) [1921] or T regulatory (Treg)-like cells (secreting IL-10, with immunosuppressive functions and probably tumor promoter activity) [2225].

Moreover, Th17 cells have the important task of coordinating the immune defense against bacterial and fungal infections and physiologically protect humans against these diseases [2628]. However, the behavior of Th17 in several tumor types and hematologic malignancies remains to be clarified [22, 2931]. In particular, different studies on AML have attributed a controversial pathogenetic role and divergent prognostic values to these cells [3136] but have not succeeded in establishing a link between the reported alterations and the infections to which these patients are subject.

We showed that Th17 cells with a double production of IL-17 and IL-10 were strongly increased in AML patients and that ex vivo patient immune response to an infectious antigen, such as Candida Albicans (C. Albicans), was significantly reduced by Th17. Finally, we found that blasts, co-cultured with CD4+ cells from healthy donors, were able to change the frequency and cytokine profile of T cells, and in particular of Th17, in a similar manner to that observed in patients.

All of the above data support the hypothesis that the increase in IL-10+ Th17 cells in AML is a mechanism developed by the disease to create an immunosuppression state which, given the stem-like features and long life of Th17 cells [14, 37], may be durable and ultimately favor infections, protecting leukemia cells from immune control.


Blood samples and PBMCs collection

After obtaining the patient’s informed consent and the approval of the local ethics committee, in accordance with the Declaration of Helsinki, samples of peripheral blood (15–20 ml) were collected from 30 newly diagnosed AML patients before any treatment was started and from 30 age-matched (±10 years) healthy volunteers (HV). AML patients were diagnosed according to the French American-British (FAB) classification system [38]. Patient and HV characteristics are reported in Table 1. Blood samples were collected in sterile EDTA tubes and mononuclear cells (PBMCs) were separated by density gradient centrifugation using Lymphosep (Biowest) and frozen in 90% heat inactivated fetal bovine serum (FBS) (PAA) and 10% dimethylsulfoxide (Sigma Aldrich).
Table 1

Patient and HV characteristics




No. patients






15 (50%)

14 (47%)


15 (50%)

16 (53%)

Median age, years (range)

63 (38–87)

68 (35–85)

Subtype according to FAB classification



6 (20%)



9 (30%)



4 (13.3%)



9 (30%)



1 (3.3%)



1 (3.3%)




14 (47%)



4 (13%)



3 (10%)

Trisomy chr 8


3 (10%)



2 (7%)

 Tetrasomy chr 21


1 (3%)

 Inv chr 3


1 (3%)

 Del chr 7


1 (3%)

 Del chr 20


1 (3%)

Molecular mutations

 FLT3/NPM wt


17 (57%)



4 (13%)

 FLT3 mut NPM mut


3 (10%)

 FLT3 mut NPM wt


1 (3%)

 FLT3 wt NPM mut


1 (3%)



2 (7%)



2 (7%)

FAB French-American-British, chr chromosome, t translocation, Inv inversion, Del deletion, wt wild type, mut mutated.

CD4+ cell isolation and culture

In order to avoid contamination by CD4+ cells that release IL-17, such as macrophages [37], PBMCs were thawed and human CD4+ T cells were isolated by negative depletion of CD8+, CD14+, CD15+, CD16+, CD19+, CD36+, CD56+, CD123+, TCR y/δ and CD235a+, using the CD4+ T cell isolation kit (Miltenyi Biotec). In this way also AML blasts, where present, were included in the subsequent analysis. Cells were cultured in RPMI 1640 medium (PAA) supplemented with 10% heat inactivated FBS, 2 mM l-glutamine (Euroclone), penicillin (100 U/ml) and streptomycin (100 μg/ml) (PAA). CD4+ cells were primed for 24 h at 37°C with IL-6 (30 ng/ml) (Miltenyi Biotec) or TGF-β (10 ng/ml) (Abcam) or a combination of IL-6 and TGF-β. T cells were then incubated for 5 h at 37°C with phorbol 12-myristate-13-acetate (PMA, 50 ng/ml) and ionomycin (1 μg/ml) (Invitrogen) in the presence of GolgiStop Protein Transport Inhibitor (BD Pharmingen). An unstimulated control prepared by incubating CD4+ cells with GolgiStop Protein Transport Inhibitor only was included for each experiment.

Immunophenotypic analysis of T cells

After stimulation, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) then immunophenotyped for intracellular IFN-γ, IL-4 and IL-17A expression using the human TH1/TH2/TH17 phenotyping kit (BD Pharmingen) following the manufacturer’s protocol. For Treg analysis, naïve PBMCs were stained with anti-human FITC CD4 (0.6 μg/ml, clone SK3; BD Biosciences) and anti-human APC-Cy7 CD25 (2.5 μg/ml, clone M-A251; BD Biosciences) for 10 min at 4°C in the dark. After incubation, cells were fixed and permeabilized and then stained with anti-human APC FoxP3 (1:11, clone 3G3; Miltenyi Biotec) for 30 min at 4°C in the dark. Appropriate isotype controls were included for each sample.

Cytokine secretion analysis

Stimulated CD4+ cells were washed with cold PBS containing 0.5% (v/v) bovine serum albumin (BSA) (Sigma Aldrich) and 2 mM of EDTA and analyzed using human IL-17 and IL-10 secretion assay—detection kits (Miltenyi Biotec). Briefly, cells were stained with IL-17 and IL-10 catch reagents for 5 min on ice, incubated for 45 min at 37°C to allow cytokine secretion and then with anti-human PE IL-17A, anti-human APC IL-10 and anti-human FITC CD4 for 10 min on ice, according to the manufacturer’s instructions. Samples were washed and suspended for flow cytometric analysis.

CD33+ cells isolation

Circulating CD33+ cells were magnetically isolated from AML PBMCs in two steps: first, CD4+ and blast cells were negatively purified using the T cell isolation kit, as already described; subsequently, CD33+ cells were purified with CD33 MicroBeads kit (Miltenyi Biotec) following the manufacturer’s instructions.

Direct and indirect allogeneic co-cultures

For direct co-cultures, CD33+ cells isolated from 15 AML patients and allogeneic CD4+ T cells obtained from 15 HV as previously reported were co-seeded in 1:1, 1:5 and 1:10 ratios. For indirect co-cultures, purified CD4+ cells were seeded in the bottom part of the 6-well plates of transwell cell culture system (pore size 0.4 μm; Costar Corp.), whereas CD33+ cells were seeded in the corresponding transwell cell culture inserts. In addition, each cell type was seeded individually in 6-well plates for single culture as control. All samples were cultured in complete medium and stimulated as previously described. At the end of stimulation, T cell immunophenotypic and cytokine secretion analysis was performed.

T cell activation with C. Albicans and isolation of IL-17-secreting cells

CD4+ cells (2.5 × 106) were stimulated for 24 h at 37°C with 1 μg/ml of C. Albicans peptides (JPT, Berlin, Germany). During the last 5 h of incubation, cells were maintained in the presence of GolgiStop Protein Transport Inhibitor (BD Pharmingen). Samples were centrifuged at 4°C, incubated with 2 mM of EDTA in PBS for 10 min at 37°C, washed with 0.5% BSA and 0.1% sodium azide in PBS. Cells were then depleted of IL-17-secreting cells using the IL-17 Secretion Assay—Cell Enrichment and Detection Kit (Miltenyi Biotec). The IL-17 specific catch reagent was attached to the cell surface as previously described, after which cells were labeled with anti-human PE IL-17A and stained with anti-PE microbeads. IL-17-secreting cells were separated through two consecutive column runs, according to the manufacturer’s instructions. Negative fraction was cultured for a further 24 h in complete medium supplemented with 1 μg/ml of C. Albicans peptides and then analyzed for intracellular IFN-γ expression using the human TH1/TH2/TH17 phenotyping kit (BD Pharmingen). A sample stimulated with C. Albicans for 48 h without depletion of IL-17-secreting cells was added as control.

Flow cytometry

Flow cytometric analysis were performed using a FACSCanto flow cytometer (Becton–Dickinson) equipped with 488 nm (blue) and 633 (red) lasers and 50,000 events were recorded for each sample. The acquisition and analysis gates were set on lymphocytes based on forward (FSC) and side scatter (SSC) properties of cells. FSC and SSC were set in a linear scale. For more extensive analysis, gates were set on CD4+ T cell subsets. Flow cytometry data were analyzed with Diva Software (Becton–Dickinson).

Statistical analysis

Data were summarized by descriptive statistics (mean ± standard deviation for continued variables and frequency and percentage for categorical variables). Statistical analyses were carried out using the paired and unpaired two-tailed Student’s t tests and confirmed with the non parametric Wilcoxon signed-rank test. P values <0.05 were considered as significant.


Alterations in the T cells frequency in peripheral blood of AML patients

We first focused on the frequency of CD4+ T cells (Th1, Th2, Th17 and Tregs) in the peripheral blood of 30 newly diagnosed untreated AML patients characterized by karyotype and molecular biology mutations (Table 1) and 30 sex- and age-matched HV. Given the controversies over the different Th17 polarization methods [3943], we stimulated CD4+ cells isolated through a negative immunomagnetic system in serum-containing media with IL-6 and TGF-β alone and in combination. As no significant differences were observed (data not shown), Th1, Th2 and Th17 analyses were performed with IL-6 alone, thereby reducing the risk of the TGF-β-mediated induction or inhibition of other cytokines.

As shown in Figure 1a, b, flow cytometric analysis revealed that the frequencies of T helper populations were altered in AML patients in comparison to healthy donors. In particular, Th1 and Th2 percentages were lower in patients (4.0 ± 2.1 and 0.8 ± 0.6% respectively) than in controls (12.8 ± 4.3 and 2.9 ± 1.8% respectively), whereas Th17 cells showed a 1.5-fold increase in AML patients compared to HV (P = 0.013) (Figure 1c). All the observed differences were statistically significant.
Figure 1
Figure 1

Alterations in T helper cells from HV and untreated AML patients. a Representative dot plots of cytokine production by CD4+ cells isolated from a HV before and after in vitro priming with IL-6 and then with phorbol 12-myristate 13-acetate (PMA) and ionomycin (I). b Representative dot plots of CD4+ cells from an AML patient before and after stimulation with IL-6 and PMA + I. c Pooled data obtained after stimulation from 30 HV (white circles) and 30 AML patients (black circles) and mean values (bars). d Gating strategy used to identify the CD4+ CD25highFoxP3+ cells. e Mean and standard deviation of Treg frequency from HV and AML patients.

In addition the frequency of circulating Tregs was performed in unstimulated PBMCs by flow cytometry. CD4+ cells with mean fluorescence intensity of CD25 expression ≥10-fold the negative cut-off were classified as CD25high as previously reported [11] (Figure 1d). Our data highlighted a significantly higher frequency of CD4+ CD25high FoxP3+ cells in AML samples (0.83 ± 0.75%) than in controls (0.19 ± 0.15%) (P = 0.012) (Figure 1e), whereas no difference were observed in the frequency of CD4+ CD25+ FoxP3+ cells (P = 0.38, data not shown).

Altered frequency and cytokine profile were already been described in AML but not for all of these subsets of CD4+ cells in the same time and anyway, may suggest a global alteration of patient immune response.

Increased IL-17/IL-10-secreting cells in the peripheral blood of AML patients

To deepen this aspect, we also evaluate the ability of the increased Th17 cells to simultaneously produce or secrete other cytokines, focusing on IFN-γ and IL-10 [21, 2325] (Figure 2a, b). Intriguingly, we observed a strong, statistically significant increase in the frequency of CD4+ IL-17/IL-10 double secreting cells in AML patients compared to HV (0.54 ± 0.63 and 0.075 ± 0.145% respectively; P = 0.002) (Figure 2c), whereas the percentage of IL-17 +/IFN-γ+ cells remained unchanged (P = 0.39) (Figure 2d). No correlation was observed with specific karyotype, molecular biology mutations or Treg frequency.
Figure 2
Figure 2

IL-10 and IFN-γ intracellular production by Th17 cells in HV and AML patients. a Representative flow cytometric data of IL-17 and IL-10 double release by CD4+ T cells from HV and AML primed with IL-6 and PMA + I. b Representative dot plots of IL-17 and IFN-γ simultaneous production from a HV and an AML patient. c IL-17/IL-10 pooled data from HV (white circles) and AML patients (black circles) and mean values (bars). The frequency of CD4+ T cells that simultaneously secreted IL-17 and IL-10 was 7.5-fold increased in AML patients compared with HV. d IL-17 and IFN-γ production data derived from the entire series are shown as mean and standard deviation The concomitant intracellular production of IFN-γ and IL-17 by CD4+ cells was not significantly different from that of HV or patients (P > 0.05).

The increase of IL-10+ Th17, together with the aforementioned global CD4+ altered frequency, taking into account the singular infectious susceptibility of AML patients and the physiological protective role of Th17 cells, seem to strengthen the hypothesis of a reduced immune response in these patients.

T cell immune response abnormalities after stimulation with C. Albicans

In order to evaluate the functional effects of the observed alterations, we analyzed intracellular IFN-γ expression of CD4+ cells from HV and AML patients stimulated with C. Albicans with or without depletion of IL-17-secreting cells (Figure 3a).
Figure 3
Figure 3

IFN-γ production by CD4+ cells stimulated with C. Albicans before and after depletion of IL-17-secreting cells. a CD4+ cells were stimulated for 24 h with peptides from C. Albicans, then IL-17- secreting cells were then immunomagnetically depleted and negative fraction was cultured for a further 24 h in the presence of C. Albicans peptides and analyzed for intracellular IFN-γ expression. Representative dot plots of CD4+/IFN-γ+ cells from HV and AML patient before and after depletion of IL-17-secreting cells are shown. b Pooled data from 5 HV and 5 AML patients are shown as mean values and standard deviation (n.s. not significant).

As shown in Figure 3b, the number of IFN-γ+ cells in not depleted control samples was 2.4-fold lower in AML compared to HV (0.5 ± 0.2 and 1.2 ± 0.6% respectively; P = 0.025). Interestingly, in patients the IFN-γ production increased after IL-17-secreting cells depletion (2.3 ± 0.7%; P = 0.0005) (Figure 3b). Conversely, in HV, the depletion of IL-17-secreting cells did not induce significant changes in the production of IFN-γ (P = 0.21).

This in vitro functional proof is in line with the hypothesis of a reduced immune response in AML caused by altered Th17 cells; on the contrary, in HV the depletion of Th17 cells did not change the IFN-γ response, probably because under physiological conditions, they are not able to make that, alone.

The impairment of T cells after direct and indirect allogeneic co-culture with CD33+ leukemic blasts

To investigate the role of leukemic cells in T cell changes, we performed direct and indirect co-cultures of CD4+ cells obtained from 15 HV and CD33+ blast cells magnetically isolated from 15 AML patients. A cytokine pattern similar to that found in AML patients was observed in CD4+ co-cultured directly at a 1:1 ratio, with a significant reduction in IFN-γ (P = 0.0008) and IL-4-positive cells (P = 3.2E−05) and a strong increase in the percentage of CD4+ IL-17A/IL-10-secreting cells (0.02 ± 0.05 and 0.56 ± 0.5% before and after co-culture respectively; P = 0.008). The above described cytokine alterations were also present when blasts and CD4+ cells were physically separated by a membrane (Figure 4a). In both co-culture methods, these alterations were not observed at CD33+ and CD4+ ratios of 1:5 and 1:10 (Figure 4b).
Figure 4
Figure 4

CD33+ blast involvement in inducing alterations in cytokine production by Th1,Th2 and Th17. a CD4+ cells isolated from 15 HV and CD33+ cells from 15 AML were co-seeded in a 1:1 ratio in the same well [direct co-culture (CC)] or in a transwell cell culture system (indirect CC). All samples were stimulated with IL-6 and PMA + I as previously described. At the end of stimulation, T cell immunophenotypic and cytokine secretion analysis were achieved. Data (mean and standard deviations) from direct or indirect CC were compared with pooled data obtained from 15 HV (*P < 0.05, **P < 0.005, ***P < 0.0005). b HV CD4+ cells and CD33+ blasts were directly or indirectly co-seeded in 1:5 and 1:10 ratios, respectively. All samples were stimulated with IL-6 and PMA + I and then analyzed for IFN-γ, IL-4 and IL-17 production and IL-17 and IL-10 simultaneous secretion. Data are expressed as mean and standard deviation (P > 0.05).

These finds suggest that the changes observed in CD4+ cells cytokine profile were directly induced by leukemic cells probably by soluble factors.

Altered T cell cytokine production after depletion of CD33+ blasts in samples from AML patients

To provide other evidence of the blast action on CD4+ cells, we characterize the pattern of cytokine expression in AMLs before and after depletion of CD33+ blasts (Figure 5a). When patient CD4+ T cells were depleted of CD33+ cells, the former regained a capacity to produce IFN-γ and IL-4 similar to that of HV (P = 0.007 and P = 0.0001, respectively) whereas Th17 cell frequency tended to decrease but not significantly probably due to the high standard deviation among samples (Figure 5b). Interestingly, IL-17/IL-10-releasing cells significantly decreased after CD33+ removal, suggesting the involvement of blasts also in maintaining the immunosuppressive state in AML patients.
Figure 5
Figure 5

CD33+ cells are required for maintaining the altered cytokine pattern produced by T cells. a The scatter plot shows the blast compartment before and after the CD33 depletion. The dot plots show how the intracellular cytokine production changes before and after blast depletion on the same patient. All samples were stimulated with IL-6 for 24 h and PMA + I for 5 h. b Pooled data are shown as mean and standard deviation (*P < 0.05, ***P < 0.0005).


AML patients at the onset of disease and during chemotherapy are at high risk of severe and potentially fatal infections [24], but such conditions cannot be attributed to neutropenia alone. Indeed, concomitant reduced immune surveillance [9, 10, 12, 13], favors this infectious trend, worsens prognosis and limits therapeutic possibilities [3]. Multiple mechanisms of immunosuppression have been identified including indoleamine 2, 3-dioxygenase [9] and CD200 glycoprotein overexpression [10], an enhanced Treg activity [11], an impaired dendritic cell maturation [12] and PD1- PDL1 axis alteration [13].

More recently, another immunosuppressive mechanism was described, attributing defective immunological synapse formation to T cells [6]. All these mechanisms cooperate to suppress immune control on leukemia cells and infections, and also reduce the effect of vaccination or other adoptive T cell transfer strategies [7]. Nevertheless, the immune system may also be effective in controlling AML, as occurs in hematopoietic stem cell transplantation [8], and exploring additional ways to use such weapon could have a strong impact on the prognosis of these patients.

To our knowledge no convincing correlation has yet been found between immunosuppression, or specific T cell dysfunction, in AML and the infections to which AML patients are susceptible. Th17 cells, a particular subset of CD4+ cells and their respective cytokines, play a pivotal role in the inflammatory response and autoimmune diseases [1518] and also direct the defense against bacterial and fungal infections of the gastrointestinal tract, skin, airways and lungs [2628]. However, their function in several tumor types is controversial [19, 21, 22], and their involvement in hematological malignancies, in particular AML, remains to be defined [2936]. Although our results confirmed previously published data [31, 3336] showing statistically higher Th17 cell percentages in the peripheral blood of newly diagnosed AML patients compared to HV, the alteration in our study was observed together with a significant reduction in Th1 and Th2 frequencies. Moreover, a substantial increase in Tregs was observed, as previously reported by Szczepanski et al. [11].

Several studies hypothesized a role for Th17 cells in the pathogenesis of AML, but conflicting data on the different prognostic significance assigned to these cells [3133] suggest an incomplete understanding of the mechanisms involved. In our opinion, the clinical presentation of AML patients, frequently affected by severe fungal or bacterial infections, is the most important event to be taken into account together with the increased percentage of Th17 cells [31, 33, 36], as confirmed in our experiments. Indeed, these two events are clearly conflicting, given the physiological role of defense of Th17 cells. For this reason, we also performed a more in-depth investigation into the ability of Th17 cells to produce IL-17 simultaneously with other cytokines, focusing our research on IL-10 [23] and IFN-γ [21]. A simultaneous production of these cytokines has already been demonstrated in Th17 [2123, 25, 44], in line with their plasticity [14], and may also be a sign of their epigenetic transdifferentiation into other T cell types, such as Tregs [22, 23], which, themselves, may differentiate into FOXP3+ IL-17A cells [44] or Th17 Th1-like cells, secreting IFN-γ [20, 21].

We observed a significantly higher increase in the frequency of CD4+ IL-17A+/IL-10+ secreting cells in AML patients than in HV, whereas the percentage of IL-17A+/IFN-γ+ cells remained unchanged. Our results thus suggest that the substantial imbalance between IL-17/IL-10-producing cells (the involvement of FoxP3+IL-17A+IL-10+ cells cannot be excluded) and IL-17A/IFN-γ-producing cells, together with a reduced frequency in Th1 and Th2 cells, may act as an additional immunosuppressive factor in these patients, altering the physiological role of Th17, contributing to the infections and probably promoting leukemia escape. Furthermore, as Th17 cells are long-lived cells with a stem-like molecular signature [37], their immunosuppressive capacity in leukemia may be powerful and more durable.

Moreover, we demonstrated that the immune response of CD4+ cells isolated from patients was strongly reduced against an infective antigen of fungal origin and, notably, that the selective depletion of Th17 cells from the culture, led to a restoration of IFN-γ production.

To investigate the role of circulating leukemic blasts in the observed alteration, we selected CD33+ cells after depletion of myeloid differentiated cells. All the changes observed in Th17 were induced in vitro by CD33+ leukemic cells, as confirmed by direct and indirect co-cultures of healthy CD4+ cells and AML peripheral blasts. Given that in both co-culture the intensity of T alterations were similar, we hypothesized that leukemic cells action was mediated by soluble factors.

Moreover, patient T cells, depleted of CD33+ blasts, regained the capacity to produce levels of IFN- γ and IL-4 similar to those of HV and showed a decreased ability to simultaneously produce IL-17 and IL-10. Therefore all of the above data suggested the involvement of blasts also in maintaining the immunosuppressive state in AML patients.


Aside from the known immunosuppressive mechanisms of AML [913], we identified a novel process induced by leukemic cells via soluble factors through which Th17 are converted into IL-17/IL-10-secreting cells, creating an environment with reduced immune control and thus probably favoring leukemia immune escape. Moreover, for the first time we can hypothesize a direct connection between the severe infective problems of AML patients and specific T cell alterations. The frequency of the immunosuppressive IL-17/IL-10-secreting cells in patients, needs more investigations but could represent a novel, simple, prognostic tool to identify the relative risk of severe infections and, perhaps, an increased risk of relapse for AML. Finally, although the role of Th17 cells in AML warrants further investigation, their long-lasting activity and plasticity could be used to convert these cells towards a tumor suppressor activity, making them a potential target for AML immunotherapy.




acute myeloid leukemia


IL-17 T helper


T regulatory

C. Albicans

Candida Albicans


healthy volunteers




peripheral blood mononuclear cells


fetal bovine serum


phorbol 12-myristate-13-acetate


bovine serum albumin




forward scatter


side scatter


Authors’ contributions

GM and SC designed the research study, analyzed the data and wrote the paper; GM, SDM, FF and SC performed the development of methodology; CP, VG, DC, MC, MBG, AL, SR, PPF, PM, PS and MT performed the acquisition of data; GM, SDM, RN, FF and SC analyzed and interpreted the data; GM, SDM, MG, GM, WZ, DA and SC wrote, reviewed, and/or revised the manuscript; SDM, RN, FF and SC administrative the technical, or material support; GM and SC performed the study supervision. All authors read and approved the final manuscript.


The authors thank Dr. Antonio Curti (Department of Hematology and Oncological Sciences ‘L. and A. Seràgnoli’, University of Bologna, 40212 Bologna, Italy) for his conceptual contribution, Dr. Linda Valmorri for drafting of documents for the Ethics Committee, Dr. Patrizia Tosi (Hematology Unit, Infermi Hospital, 47921 Rimini, Italy) for providing blood samples, Ursula Elbling for editing the manuscript.

Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

Hematology Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy
Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy
Department of Hematology and Oncological Sciences ‘L. and A. Seràgnoli’, University of Bologna, Bologna, Italy
Internal Medicine Unit, Infermi Hospital, Faenza, Italy
Haematology Unit, Santa Maria delle Croci Hospital, Ravenna, Italy
Immunotherapy Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy
Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy


  1. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62(1):10–29PubMedView ArticleGoogle Scholar
  2. Pagano L, Caira M, Rossi G, Tumbarello M, Fanci R, Garzia MG et al (2012) A prospective survey of febrile events in hematological malignancies. Ann Hematol 91(5):767–774PubMedView ArticleGoogle Scholar
  3. Estey EH (2013) Acute myeloid leukemia: 2013 update on risk-stratification and management. Am J Hematol 88(4):318–327PubMedView ArticleGoogle Scholar
  4. Romani L (2011) Immunity to fungal infections. Nat Rev Immunol 11(4):275–288PubMedView ArticleGoogle Scholar
  5. Buggins AG, Milojkovic D, Arno MJ, Lea NC, Mufti GJ, Thomas NS et al (2001) Microenvironment produced by acute myeloid leukemia cells prevents T cell activation and proliferation by inhibition of NF- kappaB, c-Myc, and pRb pathways. J Immunol 167(10):6021–6030PubMedView ArticleGoogle Scholar
  6. Le Dieu R, Taussig DC, Ramsay AG (2009) Peripheral blood T cells in AML patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts. Blood 114(18):3909–3916PubMed CentralPubMedView ArticleGoogle Scholar
  7. Martner A, Thorèn FB, Aurelius J, Hellstrand K (2013) Immunotherapeutic strategies for relapse control in acute myeloid leukemia. Blood Rev 27(5):209–216PubMedView ArticleGoogle Scholar
  8. Schmid C, Labopin M, Nagler A, Bornhauser M, Finke J, Fassas A et al (2007) Donor lymphocyte infusion in the treatment of first hematological relapse after allogeneic stem-cell transplantation in adults with acute myeloid leukemia: a retrospective risk factors analysis and comparison with other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol 25(31):4938–4945PubMedView ArticleGoogle Scholar
  9. Curti A, Trabanelli S, Onofri C, Aluigi M, Salvestrini V, Ocadlikova D et al (2010) Indoleamine 2, 3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells. Haematologica 95(12):2022–2030PubMed CentralPubMedView ArticleGoogle Scholar
  10. Tonks A, Hills R, White P, Rosie B, Mills KI, Burnett AK et al (2007) CD200 as a prognostic factor in acute myeloid leukaemia. Leukemia 21(3):566–568PubMedView ArticleGoogle Scholar
  11. Szczepanski MJ, Szajnik M, Czystowska M, Mandapathil M, Strauss L, Welsh A et al (2009) Increased frequency and suppression by regulatory T cells in patients with acute myelogenous leukemia. Clin Cancer Res 15(10):3325–3332PubMed CentralPubMedView ArticleGoogle Scholar
  12. Mohty M, Jarrossay D, Lafage-Pochitaloff M, Zandotti C, Brière F, de Lamballeri XN et al (2001) Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment. Blood 98(13):3750–3756PubMedView ArticleGoogle Scholar
  13. Zhou Q, Munger ME, Highfill SL, Tolar J, Weigel BJ, Riddle M et al (2010) Program death-1 signaling and regulatory T cells collaborate to resist the function of adoptively transferred cytotoxic T lymphocytes in advanced acute myeloid leukemia. Blood 116(14):2484–2493PubMed CentralPubMedView ArticleGoogle Scholar
  14. Muranski P, Restifo NP (2013) Essentials of Th17 cell commitment and plasticity. Blood 121(13):2402–2414PubMed CentralPubMedView ArticleGoogle Scholar
  15. Ouyang WJ, Kolls JK, Zheng Y (2008) The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 28(4):454–467PubMed CentralPubMedView ArticleGoogle Scholar
  16. Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y et al (2008) Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med 205(4):799–810PubMed CentralPubMedView ArticleGoogle Scholar
  17. Zhang H, Kong H, Zeng X, Guo L, Sun X, He S (2014) Subsets of regulatory T cells and their roles in allergy. J Transl Med 12(12):125PubMed CentralPubMedView ArticleGoogle Scholar
  18. Monteleone I, Sarra M, Pallone F, Monteleone G (2012) Th17-related cytokines in inflammatory bowel diseases: friends or foes? Curr Mol Med 12(5):592–597PubMedView ArticleGoogle Scholar
  19. Muranski P, Restifo NP (2009) Does IL-17 promote tumor growth? Blood 114(2):231–232PubMedView ArticleGoogle Scholar
  20. Zou W, Restifo NP (2010) T(H)17 cells in tumour immunity and immunotherapy. Nat Rev Immunol 10(4):248–256PubMed CentralPubMedView ArticleGoogle Scholar
  21. Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A et al (2008) Tumor- specific Th17-polarized cells eradicate large established melanoma. Blood 112(2):362–373PubMed CentralPubMedView ArticleGoogle Scholar
  22. Martin F, Apetoh L, Ghiringhelli F (2012) Controversies on the role of Th17 in cancer: a TGF-β-dependent immunosuppressive activity? Trends Mol Med 18(12):742–749PubMedView ArticleGoogle Scholar
  23. Ye J, Su X, Hsueh EC, Zhang Y, Koening JM, Hoft DF et al (2011) Human tumor- infiltrating Th17 cells have the capacity to differentiate into IFN-γ+ and FOXP3+ T cells with potent suppressive function. Eur J Immunol 41(4):936–951PubMedView ArticleGoogle Scholar
  24. Ustun C, Miller JS, Munn DH, Weisdorf DJ, Blazar BR (2011) Regulatory T cells in acute myelogenous leukemia: is it time for immunomodulation? Blood 118(19):5084–5095PubMed CentralPubMedView ArticleGoogle Scholar
  25. Zielinski CE, Mele F, Aschenbrenner D, Jarrossay D, Ronchi F, Gattorno M et al (2012) Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1β. Nature 484(7395):514–518PubMedView ArticleGoogle Scholar
  26. Khader SA, Gaffen SL, Kolls JK (2009) Th17 cells at the crossroads of innate and adaptive immunity against infectious diseases at the mucosa. Mucosal Immunol 2(5):403–411PubMed CentralPubMedView ArticleGoogle Scholar
  27. Wuthrich M, Deepe GS, Klein B (2012) Adaptive immunity to fungi. Annu Rev Immunol 30:115–148PubMed CentralPubMedView ArticleGoogle Scholar
  28. Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M et al (2006) Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 203(10):2271–2279PubMed CentralPubMedView ArticleGoogle Scholar
  29. Prabhala RH, Pelluru D, Fulciniti M (2010) Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma. Blood 115(26):5385–5392PubMed CentralPubMedView ArticleGoogle Scholar
  30. Jain P, Javdan M, Feger FK, Chiu PY, Sison C, Damle RN et al (2012) Th17 and non-Th17 interleukin-17-expressing cells in chronic lymphocytic leukemia: delineation, distribution, and clinical relevance. Haematologica 97(4):599–607PubMed CentralPubMedView ArticleGoogle Scholar
  31. Han Y, Ye A, Bi L, Wu J, Yu K, Zhang S (2014) Th17 cells and interleukin-17 increase with poor prognosis in patients with acute myeloid leukemia. Cancer Sci 105(8):933–942PubMedView ArticleGoogle Scholar
  32. Ersvaer E, Liseth K, Skavland J, Gjertsen BT, Bruserud Ø (2010) Intensive chemotherapy for acute myeloid leukemia differentially affects circulating TC1, TH1, TH17 and TREG cells. BMC Immunol 11:38PubMed CentralPubMedView ArticleGoogle Scholar
  33. Abousamra NK, Salah El-Din M, Helal R (2013) Prognostic value of Th17 cells in acute leukemia. Med Oncol 30(4):732PubMed CentralPubMedView ArticleGoogle Scholar
  34. Tian T, Yu S, Wang M, Yuan C, Zhang H, Ji C et al (2013) Aberrant T helper 17 cells and related cytokines in bone marrow microenvironment of patients with acute myeloid leukemia. Clin Dev Immunol 2013:915873PubMed CentralPubMedView ArticleGoogle Scholar
  35. Civini S, Jin P, Ren J, Sabatino M, Castiello L, Jin J et al (2013) Leukemia cells induce changes in human bone marrow stromal cells. J Transl Med 11:298PubMed CentralPubMedView ArticleGoogle Scholar
  36. Wu C, Wang S, Wang F, Chen Q, Peng S, Zhang Y et al (2009) Increased frequencies of T helper type 17 cells in the peripheral blood of patients with acute myeloid leukaemia. Clin Exp Immunol 158(2):199–204PubMed CentralPubMedView ArticleGoogle Scholar
  37. Muranski P, Borman ZA, Kerkar SP, Klebanoff CA, Ji Y, Sachez-Perez L et al (2011) Th17 cells are long lived and retain a stem cell-like molecular signature. Immunity 35(6):972–985PubMed CentralPubMedView ArticleGoogle Scholar
  38. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al (1976) Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol 33(4):451–458PubMedView ArticleGoogle Scholar
  39. Kim JS, Jordan MS (2013) Diversity of IL-17-producing T lymphocytes. Cell Mol Life Sci 70(13):2271–2290PubMed CentralPubMedView ArticleGoogle Scholar
  40. Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE et al (2010) Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling. Nature 467(7318):967–971PubMed CentralPubMedView ArticleGoogle Scholar
  41. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F (2007) Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 8(9):942–949PubMedView ArticleGoogle Scholar
  42. Yang L, Anderson DE, Baecher-Allan C, Hastings WD, Bettelli E, Oukka M et al (2008) IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 454(7202):350–352PubMed CentralPubMedView ArticleGoogle Scholar
  43. Ganjalikhani Hakemi M, Ghaedi K, Andalib A, Hosseini M, Rezaei A (2011) Optimization of human Th17 cell differentiation in vitro: evaluating different polarizing factors. In Vitro Cell Dev Biol Anim 47(8):581–592PubMedView ArticleGoogle Scholar
  44. Koenen HJ, Smeets RL, Vink PM, van Rijssen E, Boots AM, Joosten I (2008) Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 112(6):2340–2352PubMedView ArticleGoogle Scholar


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