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

Identification of a miR-146b-FasL axis in the development of neutropenia in T large granular lymphocyte leukemia

T Large Granular Lymphocytes leukemia is characterized by the expansion of several Large Granular Lymphocyte clones, among which a subset of Large Granular Lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNA has not been evaluated in T-Large Granular Lymphocyte Leukemia patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-Large Granular Lymphocyte Leukemia through an altered expression of miRNAs. The expression level of 756 mature miRNAs was assessed on purified T-LGLs by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-Large Granular Lymphocytes. Remarkably, CD8 T-Large Granular Lymphocytes exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of FasL, that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-Large Granular Lymphocytes occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-Large Granular Lymphocytes lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-Large Granular Lymphocyte Leukemia

STAT3 mutation impacts biological and clinical features of T-LGL leukemia

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features.Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8\ub1) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France).Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8\ub1 T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia

A high definition picture of somatic mutations in chronic lymphoproliferative disorder of natural killer cells

The molecular pathogenesis of chronic lymphoproliferative disorder of natural killer (NK) cells (CLPD-NK) is poorly understood. Following the screening of 57 CLPD-NK patients, only five presented STAT3 mutations. WES profiling of 13 cases negative for STAT3/STAT5B mutations uncovered an average of 18 clonal, population rare and deleterious somatic variants per patient. The mutational landscape of CLPD-NK showed that most patients carry a heavy mutational burden, with major and subclonal deleterious mutations co-existing in the leukemic clone. Somatic mutations hit genes wired to cancer proliferation, survival, and migration pathways, in the first place Ras/MAPK, PI3K-AKT, in addition to JAK/STAT (PIK3R1 and PTK2). We confirmed variants with putative driver role of MAP10, MPZL1, RPS6KA1, SETD1B, TAOK2, TMEM127, and TNFRSF1A genes, and of genes linked to viral infections (DDX3X and RSF1) and DNA repair (PAXIP1). A truncating mutation of the epigenetic regulator TET2 and a variant likely abrogating PIK3R1-negative regulatory activity were validated. This study significantly furthered the view of the genes and pathways involved in CLPD-NK, indicated similarities with aggressive diseases of NK cells and detected mutated genes targetable by approved drugs, being a step forward to personalized precision medicine for CLPD-NK patients.Peer reviewe

Identification of novel STAT5B mutations and characterization of TCR beta signatures in CD4+T-cell large granular lymphocyte leukemia

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCR beta sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.Peer reviewe

Pro-inflammatory cells sustain leukemic clonal expansion in T-cell large granular lymphocyte leukemia

T-cell large granular lymphocyte leukemia (T-LGLL) is a chronic lymphoproliferative disorder characterized by the clonal expansion of T-cell large granular lymphocytes (T-LGL). Immunophenotypic and genotypic features contribute to discriminate symptomatic (CD8+ STAT3-mutated T-LGLL) from clinically indolent patients, this latter group including CD8+ wildtype (wt), CD4+ STAT5B-mutated and wt cases. T-LGL lymphoproliferation is sustained both by somatic gain-offunction mutations (i.e., STAT3 and STAT5B) and by pro-inflammatory cytokines, but little information is available on the activity of T-LGLL non-leukemic cells. In this study, we characterized pro-inflammatory cells in the peripheral blood of T-LGLL patients and analyzed their role in supporting the leukemic growth. In symptomatic patients we found that cell populations not belonging to the leukemic component showed a discrete pro-inflammatory pattern. In particular, CD8+ STAT3-mutated cases showed a skewed Th17/Treg ratio and an abnormal distribution of monocyte populations characterized by increased intermediate and non-classical monocytes. We also demonstrated that monocytes released high levels of interleukin-6 after CCL5 stimulation, a chemokine specifically expressed only by leukemic LGL. Conversely, in asymptomatic cases an altered distribution of monocyte populations was not detected. Moreover, T-LGLL patients’ monocytes showed abnormal activation of signaling pathways, further supporting the different pathogenic role of monocytes in patients in discrete clinical settings. Altogether, our data contribute to deepening the knowledge on the different cell subtypes in T-LGLL, focusing particularly on non-leukemic cell populations and thus offering the rationale for new therapeutic strategies

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

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.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

Neutropenia and Large Granular Lymphocyte Leukemia: From Pathogenesis to Therapeutic Options

Large granular lymphocyte leukemia (LGLL) is a rare lymphoproliferative disorder characterized by the clonal expansion of cytotoxic T-LGL or NK cells. Chronic isolated neutropenia represents the clinical hallmark of the disease, being present in up to 80% of cases. New advances were made in the biological characterization of neutropenia in these patients, in particular STAT3 mutations and a discrete immunophenotype are now recognized as relevant features. Nevertheless, the etiology of LGLL-related neutropenia is not completely elucidated and several mechanisms, including humoral abnormalities, bone marrow infiltration/substitution and cell-mediated cytotoxicity might cooperate to its pathogenesis. As a consequence of the multifactorial nature of LGLL-related neutropenia, a targeted therapeutic approach for neutropenic patients has not been developed yet; moreover, specific guidelines based on prospective trials are still lacking, thus making the treatment of this disorder a complex and challenging task. Immunosuppressive therapy represents the current, although poorly effective, therapeutic strategy. The recent identification of a STAT3-mediated miR-146b down-regulation in neutropenic T-LGLL patients emphasized the pathogenetic role of STAT3 activation in neutropenia development. Accordingly, JAK/STAT3 axis inhibition and miR-146b restoration might represent tempting strategies and should be prospectively evaluated for the treatment of neutropenic LGLL patients