Dottorato in Medicina Molecolare

Contatti

Supervisore

Tesi di dottorato

INTRODUCTION

      Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults (Siegel et al., 2012)and current treatments remain unsatisfactory. Serious infections, resistance to therapy and relapse are the main causes of mortality among  patients (Pagano et al., 2012; Estey, 2012; Romani et al., 2011). Modulation of the immune system can help to control AML, as occurs in hematopoietic stem cell transplantation (Schmid et al., 2007) and a large percentage of patients who are probably destined to die from severe infections, could be saved by a better understanding of immunosuppressive mechanisms working in the disease and by their complete restoration. T_H17 cells and their respective cytokines play a key role in the inflammatory response and autoimmune disorders (Ouyang et al., 2008; Luger et al., 2008; Monteleone et al., 2012), and also coordinate the defense against bacterial and fungal infections (gastrointestinal tract, skin, airways and lung) (Khader et al., 2009). Moreover, the remarkable epigenetic plasticity of T_H17 cells in tumors has recently been demonstrated, together with their ability to transdifferentiate into T helper 1-like cells (secreting IFN-g and endowed with tumor suppressor activity) (Muranski and Restifo, 2009; Zou and Restifo, 2010) or into T regulatory (Treg)-like cells (secreting IL-10, probably with immunosuppressive functions and tumor promoter activity) (Martin et al., 2012; Zielinski et al., 2012; Ye et al., 2011; Ustun et al., 2011; Koenen et al., 2008). Furthermore, T_H17 cells are long-lived cells with a stem-like molecular signature (Muranski et al., 2011) and so their suppressor or promoter capacity against tumors may be powerful and more durable (Muranski et al., 2008). However, their real role in several tumor types is controversial (Muranski et al., 2009; Zou et al., 2010; Martin et al., 2012). In particular, their involvement in hematological malignancies, especially AML, and in the infections (especially fungal) to which AML patients are susceptible, remains to be defined (Wu et al., 2009; Tian et al., 2013; Ersvaer et al., 2010).

METHODS

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 without infections 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 (Bennett et al., 1976). 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).

CD4+ cell isolation and culture. PBMCs were thawed and human CD4+ T helper cells were isolated by negative depletion of CD8+, CD14+, CD15+, CD16+, CD19+, CD36+, CD56+, CD123+, TCR y/d and CD235a+, using the CD4+ T cell isolation kit (Miltenyi Biotec). 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 (25 μg/ml) (Miltenyi Biotec) or TGF-b (10 ng/ml) (Abcam) or a combination of IL-6 and TGF-b. 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) after which then immunophenotyping for intracellular IFN-γ, IL-4 and IL-17A expression was performed using the human T_H1/T_H2/T_H17 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.

Direct and indirect allogeneic co-cultures. For direct co-cultures, leukemic CD33+ blast cells magnetically isolated with CD33 MicroBeads (Miltenyi Biotec) 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.

Immunofluorescence assays. T cells (1 x 10⁶) isolated from healthy donors were stained with Cell Tracker Blue CMAC (7-amino-4 chloromethylcoumarin) (Molecular Probes) for 45 min at 37°C following the manufacturer’s instruction. CD4+ cells were co-seeded with an equal number of CD33+ cells isolated from AML patients for 24 h at 37°C and then fixed for 15 min at room temperature with 2% formaldehyde in PBS. Cells were permeabilized and stained with 1:40 Phalloidin Alexa Fluor 594 (Life Technologies) in Inside Perm (Miltenyi Biotec) for 20 min at room temperature.After labeling, specimens were washed and mounted on Prolong Gold Antifade Reagent (Invitrogen). The specificity of staining was optimized using isotype-control antibody. Fluorescence was acquired with a Evos FL Microscope (AMG, Washington, USA) equipped with Light Cube for DAPI, GFP and RFP.

T cell activation with C. Albicans and isolation of IL-17-secreting cells. CD4+ cells (2.5 x 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 2mM 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 1μg/ml of C. Albicans peptides and then analyzed for intracellular IFN-γ expression using the human T_H1/T_H2/T_H17 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.

RESULTS AND DISCUSSION

      In order to examine the relationship between T_H17 cells, leukemic cells, infections and immunocompetence, we first focused on the frequency of CD4+ T cells (T_H1, T_H2, Tregs and T_H17 cells) in the peripheral blood of 30 newly diagnosed, untreated AML patients and 30 sex- and age-matched  (+/- 10 years) healthy volunteers (HV). AML patients were diagnosed according to the French American-British classification system (Bennett et al., 1976)and characterized by karyotype and molecular biology mutations (Table 1). Given the controversies over the different T_H17 polarization methods (Ghoreschi et al., 2010; Acosta-Rodriguez et al., 2007; Yang et al., 2008; Ganjalikhani et al., 2011), we stimulated CD4+ cells isolated through a negative immunomagnetic system in serum-containing media with IL-6, TGF-β or  IL-6+TGF-β. As no significant differences were observed (Fig. S1), T_H1, T_H2 and T_H17 analyses were performed with IL-6 alone, thereby reducing the risk of the TGF-b-mediated induction or inhibition of other cytokines (Ghoreschi et al., 2010; Acosta-Rodriguez et al., 2007; Yang et al., 2008; Ganjalikhani et al., 2011). The frequency of circulating Treg cells was assessed in unstimulated PBMCs.

      Flow cytometric analysis revealed that the frequencies of T CD4+ populations were altered in AML patients (Fig.1, A-C). In particular, CD4+ cells showed a decreased capacity to produce IFN-g and IL-4, whereas the frequency of IL-17+ cells was higher in AML patients than in HV. In addition, the percentage of circulating CD4+ CD25^highFoxP3+ Tregs was higher in AML patients (Fig. 1 D-E; Fig. S2). All the observed differences were statistically significant. However, in view of the singular infectious susceptibility of these patients and to the physiological protective role of T_H17 cells, we also decided to evaluate the ability of T_H17 cells to simultaneously produce or secrete other cytokines, focusing on IL-10 and IFN-γ(Zielinski et al., 2012; Ye et al., 2011). Intriguingly, we observed a strong, statistically significant increase in the frequency of CD4+ IL-17A/IL-10-secreting cells in AML patients compared to HV (0.6±0.5 and 0.075±0.6 respectively; Fig. 1, F-G), whereas the percentage of  IL-17A+/IFN-γ+ cells remained unchanged (Fig. S3). No correlation was observed with specific karyotype or molecular biology mutations. Having identified an alteration that probably leads to an immunosuppressed state, we proceeded to confirm this and to evaluate its effect on the immune response. We stimulated CD4+ cells from HV and untreated AML patients with an infectious fungal antigen from Candida Albicans (C. Albicans). The samples were then depleted of IL-17-secreting cells, cultured for a further 24 h with C. Albicans peptides and analyzed for intracellular IFN-γ expression. Other samples from HV and AML patients stimulated with C. Albicans as above but not depleted of IL-17-secreting cells were used as control. A significant reduction in the frequency of IFN-g positive cells was observed in control samples from AML patients compared to HV. Interestingly, after removal of the IL-17-secreting cells, the IFN-g production has returned elevated in patients (Fig. 2, A-B). Conversely, in HV, the depletion of IL-17-secreting cells did not induce significant changes in the production of IFN-g.

      Finally, to investigate the role of leukemic cells in such changes, we performed direct and indirect co-cultures of CD4+ T cells obtained from 15 HV and CD33+ blast cells magnetically isolated from 15 AML patients. After 24 h, T cell immunophenotypic and cytokine secretion analyses were performed. A cytokine pattern similar to that found in AML patients was observed in CD33+ and CD4+ co-cultured directly at a    1:1 ratio, with a reduction in IFN-g and IL-4-positive cells and a strong increase in the percentage of CD4+ IL-17A/IL-10-secreting cells (Fig. 3 A). Moreover, cells stained with fluorescent phalloidin revealed a cellular distribution of F-actin and, in particular, cytoskeletal remodeling at the point of contact between blast and T cells, suggesting a role of cell-cell contact in the changes (Fig. 3 B). However, the observed cytokine alterations were also present when blasts were physically separated from CD4+ cells by a membrane (Fig. 3 A). In both co-culture methods, the described alterations were not observed at CD33+ and CD4+ ratios of 1:5 and 1:10 (Fig. S4), indicating that, in vitro, the number of CD33+ circulating blasts played an important role in inducing morphological and functional alteration in T helper cells. Notably, when patient CD4+ T cells were depleted of CD33+ blasts, the former regained a capacity to produce IFN-g and IL-4 similar to that of HV (Fig. 3, C-D). Furthermore, IL-17/IL-10 producing cells significantly decreased after CD33+ removal, indicating that blasts play a part in the control of this population.

      In conclusion, in addition to the known pathologic mechanisms of AML (Curti et al., 2010; Zhou et al., 2010; Szczepanski et al., 2009; Mothy et al., 2001) and to the changes in Tregs (Ustun et al., 2011)which differentiate into FOXP3+ IL-17A-secreting cells under specific stimuli (Koenen et al., 2008), our results identified a novel process in this disease that induces a substantial percentage of T_H17 cells to change into IL-17/IL-10-secreting cells with immunosuppressive activity. We also observed a dual mechanism of action mediated by contact and soluble factors that can be held responsible for these changes. Furthermore, the immunosuppressive action of T_H17 cells observed in our study would seem to lead to an alteration in the physiological role of these cells in AML patients, favoring infections and probably promoting immune escape of the disease. Further studies are needed, nevertheless the long-lasting activity and plasticity of T_H17 cells and the opportunity to restore and to change them in tumor suppressor cells, makes T_H17 an optimal new target to enhance immunotherapy in AML.

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