Pathogenesis of Neutropenia in large granular lymphocyte leukemia: immunophenotypic and molecular characterization of neutropenic patients

Large Granular Lymphocytes (LGLs) leukemia (LGLL) is a chronic lymphoproliferation of clonal cytotoxic LGLs, which can be divided in two different subsets, based on Cluster of Differentiation (CD)-3 expression: CD3- Natural Killer (NK) cells and CD3+/CD8+/CD4- or CD3+/CD8-/dim/CD4+ T-LGLs. Leukemic LGLs are characterized by the up-regulation of several pro-survival signaling pathways. Among these, the most relevant is the JAK-STAT axis, whose constitutive activation is in part explained by somatic activating mutations in STAT3 and STAT5b. Neutropenia is the most frequent clinical manifestation, but its pathogenesis is still unknown, although high levels of soluble Fas Ligand (sFasL) were detected in serum of LGLL patients and supposed to trigger neutrophil’s death. This work aimed i) to describe the distinctive biological features of T-LGLs or NK cells of neutropenic LGLL patients; ii) to investigate the role of microRNAs in the regulation of sFasL expression in leukemic LGLs. LGLs were purified by FACSAriaIII cell sorter from peripheral blood mononuclear cells (PBMCs) of untreated LGLL patients and their immunophenotype was evaluated by Flow Cytometry. The screening of STATs mutations was performed by Sanger sequencing. PBMCs of patients were cultured in vitro and STAT3 transcriptional activity were inhibited or stimulated with Stattic or IL-6, respectively. Transcriptional and protein expression levels were evaluated by Real Time-PCR, Western Blot (WB) assays and ELISA test.High throughput and single miRNA analysis were carried out on purified T-LGLs by using the TaqMan® Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. We showed that leukemic T-LGLs of neutropenic patients were characterized by CD3+/CD8+/CD16+/CD56- phenotype, higher STAT3 activations, higher incidence of STAT3 mutations and higher levels of sFasL, compared to T-LGLs of non-neutropenic patients. We also demonstrated that FasL transcription was mediated by STAT3 activation in T-LGLL patients. The characterization of neutropenic CLPD-NK patients, instead, showed that they are characterized by CD56-/dim/CD16high/CD57- cytotoxic NK cells expansion. However, we observed a heterogeneous level of STAT3 activation and a heterogeneous expression of FasL in this subset of patients. To investigate whether STAT3 could play its pathogenetic role in T-LGLs through an altered expression of microRNAs, we studied miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC). We showed that miR-146b expression, found down-regulated in neutropenic patients, was correlated with the ANC of T-LGLL patients. To investigate miR-146b role in neutropenia development, we transfected purified T-LGLs with a miR-146b mimic. We showed that restoration of miR-146b led to a decrease of FasL mRNA, without changes in the FasL primary transcript as compared to control. However, FasL was not identified among the putative miR-146b target genes, suggesting that miR-146b could regulate FasL expression indirectly. Therefore, we checked for genes involved in mRNA stability and we found that the defective miR-146b expression lead to increased transcriptional levels of the mRNA stabilizer HuR, that is required for FasL expression in T-lymphocytes10. Consistently, by WB assays, we demonstrated that in T-LGLs of neutropenic patients HuR endogenous protein levels were higher than in T-LGLs of non-neutropenic ones. HuR-mediated FasL mRNA stabilization explained the increased FasL expression observed in neutropenic patients. In this work we identify distinctive features of neutropenic LGLL patients, that could acquire relevance to correctly address the management of each patient, and we identify a miR-146b-FasL axis involved in neutropenia development in leukemic T-LGLs.