Page 192 - Haematologica May 2020

B. Mariotti et al.
ease in LGLL patients, often requiring specific therapy. The pathogenesis of neutropenia, although not properly elucidated thus far, seems to be multifactorial. Multiple mechanisms have been postulated, including destruction of mature neutrophils and myeloid progenitors via Fas/Fas ligand (FasL). Mechanisms leading to neutropenia are not completely characterized, although we5 and others9-12 pro- vided evidence that extremely high concentrations of FasL are detectable in T-LGLL patients suggesting that soluble FasL is involved in this process. We also demonstrated that STAT3 plays a central role in FasL transcription and we showed that high levels of STAT3 activation correlated with high levels of FasL expression in T-LGLL patients.5 However, the molecular mechanism through which STAT3 regulates FasL production in these patients has not yet been clarified.
Although STAT3 is an inducer of transcription of a large number of oncogenes, its relationship with microRNAs (miRNAs) has not yet been evaluated in T-LGLL patients. As a matter of fact, several miRNAs contribute to normal hematopoietic processes and many miRNAs act both as tumor suppressors and oncogenes in the pathogenesis of hematological malignancies, including acute and chronic leukemias and lymphomas, where they contribute to lym- phomagenesis acting in various cellular functions, such as the regulation of cell survival and proliferation.13,14
In this study a high throughput quantitative and qualita- tive analysis of the miRNA expression profile in leukemic LGLs was performed with the aim to investigate the role of miRNAs in clinical and biological features of T-LGLL, thus playing a role in the pathogenesis of neutropenia in T-LGLL patients.
Methods
Patients
Thirty T-LGLL patients and nine age and sex-matched healthy donors were enrolled in the study. Clinical and biological charac- teristics of patients are summarized in the Online Supplementary Table S1. At the time of the study, no patients had received treat- ment, with a follow-up ranging from 1 to 16 years. LGL prolifera- tion ranged from 48% to 91% of the lymphocyte pool. T-LGLL clonality was demonstrated as previously reported.15 The Padova Institutional Review Board approved this study and written informed consent in accordance with the Declaration of Helsinki was obtained by each subject.
Cell isolation and culture
T-LGLs were purified from peripheral blood mononuclear cells (PBMC) using the anti-CD57 microbeads (Milteny Biotec), as pre- viously reported.16 CD8+CD57+ cells used as control, were obtained from PBMCs of healthy donors by the FACSAria cell sorter (BD biosciences). Purity and viability of cell preparation were both >95%. The LGL phenotype was assessed by flow cytometry. In selected experiments PBMC from T-LGLL patients (2×106 cells/mL) were cultured in RPMI1640 (EuroClone) supple- mented with 10% FCS (Sigma-Aldrich), 2mM Gln 25mM Hepes, 100 U/mL penicillin and 100 mg/mL streptomycin (EuroClone) in the presence of 2.5 mM 5-aza-2’-deoxycytidine (DAC, Sigma- Aldrich) for three days, 15 mM STATTIC (Shelleckchem) for 24 hours (h) or DMSO (Sigma-Aldrich) as control.
miRNA and Gene-Expression Analysis
RNA was purified from CD57+ cells using the miRCURY RNA
Isolation Kit (Exiqon) according to the manufacturer’s instructions. High throughput and single miRNA expression were analysed using the TaqMan® Human microRNA Array (Card Set v3.0, Applied Biosystems) and the TaqMan® microRNA Human Assays (Applied Biosystems), respectively, as previously described.17 Expression of DNMT1, FasL, FasL-Primary Transcript (PT), HuR, GAPDH, pri-miR-146b and RPL32 mRNA was quantified by real- time quantitative PCR (RT-qPCR) as previously described,18 using specific primer pairs listed in the Online Supplementary Table S2.
Methylated DNA Immunoprecipitation (meDIP) assay
Genomic DNA was purified from CD57+ T-LGLs with the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen), according to the manufacturer’s instructions and methylated DNA Immunoprecipitation (meDIP) assay was performed as reported by Mohn et al.19 with minor modifications. The methylation levels of miR-146b promoter was analysed by qPCR using specific primers (Invitrogen, Thermo Scientific) listed in the Online Supplementary Table S2.
Western blot
T-LGLs (2.5x105) and Jurkat cells were lysed in Sample Buffer (40mM tris (hydroxymethyl) aminomethane HCl pH6.8, 7.5% glycerol, 1% sodium dodecyl sulphate). Total cell lysates were resolved on SDS-PAGE and transferred onto nitrocellulose (Millipore). The blots were incubated with specific antibodies and detected using the ImageQuant LAS 500 or the Odyssey infrared imaging system (LI-COR Biosciences).
Cell transfection
Jurkat cells (106) or freshly purified CD57+ T-LGL (107) were transfected with the indicated amount of specific oligonucleotide using the Amaxa Nucleofector (Lonza) and the Ingenio Electroporation Solution (Mirus Bio).
Enzyme-linked immunosorbent assay (ELISA)
Secreted FasL in plasma were determined by ELISA kit (RayBiotech, Georgia, USA), following the manufacturer’s recom- mendations.
Statistics
Statistical evaluation was performed by using the Mann- Whitney U-test, the Wilcoxon signed-rank test or Student t-test, as appropriate, with α set to 0.05. Correlation coefficient was deter- mined using the Spearman Rank Correlation.
For detailed information on the materials and methods used, see the Online Supplementary Material and Methods.
Results
Characterization of T-LGL miRNome
To identify miRNAs potentially involved in T-LGLL, the miRNA pattern of expression was investigated in CD57+ cells purified from six patients affected by T-LGLL and three healthy donors, using Taq-Man Human microRNA Array. Unsupervised Hierarchical Clustering Analysis (HCA) of normalized array data led to the identification of two clusters: cluster A including LGL1, LGL3, LGL5 and LGL9, and cluster B including LGL10, LGL17 together with the three healthy donors (HD1, HD2, HD4), (Figure 1A). Interestingly, according to the characterization of the T-LGLL patients enrolled in the study, LGL samples in cluster A share the CD4–CD8+CD16+CD56–CD57+ (CD8 T-LGLs) immunophenotype, while LGLs in cluster B share
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