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

Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates

International audienceIn cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease

New susceptibility loci associated with kidney disease in type 1 diabetes

WOS:000309817900008Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ∼2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2×10(-8)) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0×10(-9)). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-β1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1×10(-7)), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.Peer reviewe

Picturing Polarized Myeloid Phagocytes and Regulatory Cells by Mass Cytometry

International audienceThe immune monocyte/phagocyte system (MPS) includes numerous cell subsets of the myeloid lineage including monocyte, macrophage, and dendritic cell (DC) populations that are heterogeneous both phenotypically and functionally. Previously, we characterized these diverse MPS phenotypes with multi-parametric mass cytometry (CyTOF). In order to expansively characterize monocytes, macrophages, and dendritic cells, a CyTOF panel was designed to measure 35 identity-, activation-, and polarization-markers. Here we provide a protocol to define a reference map for the myeloid compartment, including sample preparation, to produce reference cell subsets from the monocyte/phagocyte system. In particular, we focused on monocyte-derived macrophages that were further polarized in vitro with cytokine stimulation (i.e., M-CSF, GM-CSF, IL-4, IL-10, IFNγ, and LPS), as well as monocyte-derived DCs, and myeloid-derived suppressor cells (MDSCs), generated in vitro from human bone marrow and/or peripheral blood

Differential desensitization of human δ-opioid receptors by peptide and alkaloid agonists

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Dissecting Complex Cellular Systems with High Dimensional Single Cell Mass Cytometry

International audienceThe following sections are included: Introduction ; The Mononuclear Phagocyte System ; Revisiting the Mononuclear Phagocyte System with High Dimensional Single Cell Analysis ; Future Directions ; Reference

Integration of Immature Granulocytes Quantification With the Version 2.0 UniCel DxH 800 in the HematoFlow Strategy

International audienceOBJECTIVES: Our aim was to define whether the early granulocyte cell marker (EGC%_DxH) parameter might replace immature granulocytes counts obtained by HematoFlow (IG%_HF) and/or manual differential count (IG%\ₘanual). METHODS: We conducted a study over a 10-day period in February 2014 whereby 402 samples were analyzed for the IG flag. We correlated the EGC%_DxH vs IG%_HF and IG%\ₘanual, identified any discrepant results and finally looked at the impact on our workflow by incorporation of the EGC% into our WBC differential algorithm. RESULTS: On an initial training set, a receiver operating characteristic (ROC) curve analysis showed a threshold of 0.9% for EGC%_DxH (sensitivity of 91.7%, specificity of 93.5% and an area under the curve of 0.965). Further analysis of the dataset (259 samples) found a correlation of the EGC%_DxH to all our IG% counting methods (r = 0.963). Incorporation of the EGC%_DxH into the WBC HematoFlow differential resulted in a 36% reduction of samples requiring HematoFlow and/or slide review. CONCLUSIONS: The EGC% generated by the DxH 800 can be easily incorporated into existing HematoFlow and slide review algorithms

Evaluation of whole-blood conservation reagents for Hematoflow-based WBC differential count: Unsatisfactory results

International audienceBackground:As conservation of whole blood samples undergoing white blood cell (WBC) differential performed by flow cytometry (Hematoflow) is needed, we evaluated the effects of two commercially available fixatives, namely TransfixTM and Streck Cell PreservativeTM.Methods:We focused on 15 normal samples and on 13 various pathological samples. We compared the two fixatives and cold- or room- temperature effects on various parameters provided by the Hematoflow system.Results:We observed that, even after 2 hours of sample treatment, the conservative methods led to significant modifications of the cell percentages due to substantial variations of the epitope expression.Conclusion:None of the different conservation methods is really reliable for WBC differential performed by flow cytometry and thus samples should be analyzed promptly or stored at 4°C

Regulatory myeloid cells: an underexplored continent in B-cell lymphomas

International audienceIn lymphomas arising from the germinal center, prognostic factors are linked to the myeloid compartment. In particular, high circulating monocyte or myeloid-derived suppressor cell counts are associated with poor prognosis for patients with high-grade B-cell lymphomas. Macrophages with an M2 phenotype are enriched within lymphoma tumors. However, the M1/M2 nomenclature is now deprecated and the clinical impact of this phenotype remains controversial. Across cancer types, myeloid cells are primarily thought to function as immune suppressors during tumor initiation and maintenance, but the biological mechanisms behind the myeloid signatures are still poorly understood in germinal center B-cell lymphomas. Herein, we describe the role and clinical relevance of myeloid cells in B-cell lymphoma and propose innovative approaches to decipher this complex cellular compartment. Indeed, characterization of this heterogeneous cell ecosystem has been largely accomplished with "low-resolution" approaches like morphological evaluation and immunohistochemistry, where cells are characterized using a few proteins and qualitative metrics. High-resolution, quantitative approaches, such as mass cytometry, are valuable to better understand myeloid cell diversity, functions, and to identify potential targets for novel therapies