Journal: Cancer Discovery

Article Title: Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia

doi: 10.1158/2159-8290.CD-22-1396

Figure Lengend Snippet: Divergent IL3Rα and βc expression preferentially occurs at the apex of the hematopoietic cell hierarchy and is linked to poor patient survival in AML. A, IL3Rα and βc gene expression ratio in 451 de novo normal karyotype patients with AML, as shown in reads per kilobase per million mapped reads (RPKM; Beat AML; ref. ). Av, average. B, Kaplan–Meier overall survival curve for normal karyotype patients with AML comparing those with higher or lower than 1.1 ratio of IL3Rα/βc expression in The Cancer Genome Atlas cohort ( n = 184; ref. ). C, Single-cell gene expression of IL3Rα and βc across 11,641 single AML cells from 12 patients , with cell types annotated from . Mean gene expression is depicted for each AML cell type. D, Hierarchical clustering of primary AML patient samples with high ( n = 5) and low ( n = 5) IL3Rα/βc ratio [RNA sequencing (RNA-seq)] combining transcriptional (*; RNA-seq) and immunophenotypic (flow cytometry) profiling in the Toronto cohort. E, IL3Rα and βc gene expression in sorted and functionally validated immunophenotypic cellular fractions from CB . CMP, common myeloid progenitors; GMP, granulocyte-monocyte progenitors; Gr, granulocytes; HSC, hematopoietic stem cells; MEP, megakaryocyte-erythroid progenitors; MLP, multilymphoid progenitors; Mono, monocytes; MPP, multipotent progenitors. F, Flow cytometric analysis for %IL3Rα hi βc lo population of high ( n = 5, blue) and low ( n = 5, red) IL3Rα/βc transcript ratio AML patient samples (CD3 − CD19 − CD45 + ) in the Toronto cohort based on cell-surface IL3Rα and βc protein expression profiles. CB mononuclear cells ( n = 3) and mobilized peripheral blood (mPB) mononuclear cells ( n = 1) served as controls (ctrl). ns, not significant. G, Correlation of relative abundance of cells with quiescent and primed combined transcriptional phenotypes with %IL3Rα hi /βc lo population in CD3 − CD19 − CD45 + cells with high and low IL3Rα/βc ratio (by RNA-seq) in the Toronto cohort by Pearson analysis. H, Spearman correlation between gene expression and LSC frequency from 88 AML fractions in which specific LSC frequencies were calculated by limiting dilution analysis (LDA) in xenograft assays . IL3Rα ( IL3RA ) and βc ( CSF2RB ) are highlighted. I, IL3Rα/βc transcript ratio from 138 LSC + (engrafting) and 82 LSC − (nonengrafting) fractions . J, Sorting gates for xenotransplanted high/medium (med)/low IL3Rα/βc ratio (CD123 vs. CD131) fractions for patient sample AML#140005 and AML#130578 are shown pregated for viable (SytoxBlue − ) and CD45 + CD3 − CD19 − cells and in relation to an in parallel stained G-CSF mobilized peripheral blood control sample (mPB ctrl) from a healthy donor. K, LSC frequencies of high/med/low IL3Rα/βc ratio fractions from AML#140005 and AML#130578 xenotransplanted in limiting dilution into NSG-SGM3 and NSG mice as estimated from CD45 + CD33 + engraftment (>0.1%) at 7 to 8 weeks after transplantation (see also Supplementary Fig. SIQ). Due to low cell numbers retrieved, the low-ratio fraction of AML#130578 was transplanted only into NSG-SGM3. Open triangle and dotted error bar line indicate an estimated 1/LSC frequency and upper limit of 1. Upper and lower estimated limit (error bars) and P values as calculated by ELDA.

Article Snippet: The QuantiBRITE assay was performed according to the manufacturer's instructions using anti–CD131-PE or anti–CD123-PE costained with anti–CD34-APC-Cy7 (BD, custom-made) and anti-CD38-BV711.

Techniques: Expressing, RNA Sequencing Assay, Flow Cytometry, Staining, Transplantation Assay

Journal: PLoS ONE

Article Title: BCR-ABL Affects STAT5A and STAT5B Differentially

doi: 10.1371/journal.pone.0097243

Figure Lengend Snippet: (A) TonB cells cultured in the presence of IL-3 or doxycycline were spun down on glass slides, fixed by ice-cold methanol and stained sequentially with anti-STAT5B, anti-rabbit-FITC and DAPI. Intracellular localization was analyzed using confocal microscopy. Nuclear localization was confirmed by a DNA-specific DAPI counterstain. The lower panel shows antibody (with and without enhancement) and DAPI control. (B) TonB cells expressing STAT5A-eGFP were cultured in the presence of IL-3, BCR-ABL or BCR-ABL plus imatinib mesylate (BCR-ABL+IM) and live-cell analysis was performed by confocal microscopy (upper panel). The middle panel shows STAT5A-eGFP in BCR-ABL-expressing TonB cells after removal of imatinib mesylate (−IM) for the indicated periods of time. The lower panel shows empty vector control (eGFP). (C) TonB-STAT5A-eGFP cells treated as in , middle panel, were fixed 45 minutes after imatinib-removal, stained with anti-CD131-PE antibody (IL-3/IL-5/GM-CSF-receptor common β-chain) and DAPI before analysis by confocal microscopy.

Article Snippet: Cells were blocked in PBS supplemented with 10% [v/v] FCS (Biochrome, Berlin, Germany) for 1 hour and stained with PE Rat anti-mouse CD131 (559920) from BD Pharmingen and DAPI (0.5 µg/mL) for 2 hours.

Techniques: Cell Culture, Staining, Confocal Microscopy, Expressing, Plasmid Preparation

Journal: medRxiv

Article Title: T cell anergy in COVID-19 reflects virus persistence and poor outcomes

doi: 10.1101/2020.09.21.20198671

Figure Lengend Snippet: a-d Whole blood from a healthy donor was cultured in duplicates for 24 hours at 37°C with or without cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, IL-15, GM-CSF, IFN-γ, IFN-α and TNF-α, 20 ng/ml each). Samples were analyzed by flow cytometry and absolute values of indicated cell surface markers are depicted as mean fluorescence intensity (MFI). a Expression of CD131 on basophils. b Expression of CD131 on pDCs. c Expression of CD123 on CD14+ monocytes. d Expression of CD11b on neutrophils. Data represent mean +/- standard deviation. Statistical differences between medium control and the various cytokines were calculated by one-way ANOVA with Bonferroni multiple comparison test ( * p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Anti-coagulated fresh blood samples (100 µl) or cells after whole blood stimulations were incubated with various panels of the following directly labeled monoclonal antibodies for 20 minutes by 4°C: anti-CD3 APC-Cy7 (clone: SK7, BioLegend), anti-CD4 V500 (clone RPA-T4, BD Bioscience), anti-CD8 APC-eFluor 780 (clone: RPA-T8, Invitrogen, Darmstadt, Germany) and anti-CD8 PE-Cy7 (clone: SK1, BioLegend), anti-CD11b PE-Cy7(clone: M1/70, BioLegend, San Diego, CA), anti-CD14 V500 (clone: MFP9, BD Bioscience), anti-CD16 Pacific Blue (clone: 3G8, BioLegend), anti-CD19 Pacific Blue (clone: HIB19, BioLegend), anti-CD25 APC (clone: BC96, BioLegend), anti-CD116 FITC (clone: 4H1, BioLegend), anti-CD131 PE (clone: 1C1, eBioscience), anti-CD123 PE-Cy5 (BD Bioscience), anti-CD169 PE (clone: 7-239, Miltenyi Biotec, Bergisch Gladbach, Germany), CD304 APC (clone: 12C2, BioLegend), HLA-DR II APC and FITC (clone: G-46-6, BD Bioscience).

Techniques: Cell Culture, Flow Cytometry, Fluorescence, Expressing, Standard Deviation