Patient samples
Human peripheral blood (PB) and bone marrow (BM) samples were collected from patients with hypereosinophilia (Supplementary Table 1 and 2, HES n = 18, CEL-NOS n = 9, L-HES n = 2, MLN-Eo n = 2, EGPAanca− n = 11, reactive eosinophilia n = 3) and healthy donors (n = 13) according to the institutional guidelines and in concordance with the Declaration of Helsinki. Healthy PB control samples were taken from volunteers at the TUM. Healthy BM control samples were collected from human femoral heads discarded after hip joint surgery. All subjects gave written informed consent. Clinical data of the subjects including treatment status can be found in the Supplementary (Supplementary Table 1). Patients on or after tyrosine kinase inhibition therapy have not been enrolled in this study.
Cell culture
Granulocytes from PB and BMMCs were purified using Biocoll Separation Solution 1.077 g/ml (Biochrom, Berlin, Germany). Eosinophils were purified by negative selection using CD16 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Granulocytes were cultured at a density of 5 × 105 cells/ml media containing RPMI 1640 (Life Technologies, Carlsbad, USA), 20% FBS Good Forte (PAN Biotech, Aidenbach, Germany), 25 mM HEPES, 2 mM L-glutamine, 1 × MEM NEAA, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol (all from Life Technologies, Carlsbad, USA) and 10 ng/ml rhIL-5 (Biolegend, San Diego, USA). BMMCs were kept at a density of 5 × 105 cells/ml in media containing 20% BIT 9500 (StemCell, Vancouver, Canada), 100 ng/ml rhSCF, 5 ng/ml interleukin-6, 10 ng/ml interleukin-3, 10 ng/ml thrombopoietin (all from R&D Systems, Minneapolis, USA), 10 µM β-mercaptoethanol and 4 µg/ml low-density lipoproteins (StemCell, Vancouver, Canada).
Inhibitors
ABT-199 (Abbvie, North Chicago, USA), ABT-737 (Active Biochem, Maplewood, USA), S63845 (Active Biochem, Maplewood, USA) and WEHI-539 (Aobious, Gloucester, USA) were dissolved in dimethyl sulfoxide (DMSO) and used in a final concentration of 1 µM. DMSO was used at 0.001% as soluble control.
Cell viability assay
PB granulocytes were stained with anti-human Siglec-8 (APC, Clone 7C9; Miltenyi Biotec, Bergisch Gladbach, Germany), BMMCs were stained with CD34 (eFluor 450, Clone 4H11) and CD45 (APC, Clone 2D1; both: Invitrogen, Carlsbad, USA). Viability was measured by flow cytometry of Annexin V (FITC) and 7-aminoactinomycin D (7AAD; PerCP; both Invitrogen, Carlsbad, USA). Viable cells were negative for Annexin and 7AAD. Cytometry was performed on a FACS Canto II (BD Bioscience, Franklin Lakes, USA), data were analyzed using FlowJo Version 10.5.3 (FlowJo, Ashland, USA).
Colony formation assay
After 72 h-treatment, 2 × 104 BMMCs were seeded in MethoCult (H4435, StemCell, Vancouver, Canada) in duplicates. After 14 days, numbers of granulocyte–macrophage progenitor colonies (CFU-GM) were assessed manually by microscopy.
RT-qPCR/ELISA
RNA from eosinophils was purified using an RNA isolation kit (Macherey–Nagel, Düren, Germany). RNA concentrations were normalized, cDNA was synthesized using the QuantiTect Reverse Transcription System (Qiagen, Venlo, Netherlands) in a Biometra TAdvanced Thermocycler (Analytic Jena, Jena, Germany). cDNA was analyzed with GoTaq qPCR Mastermix (Promega, Fitchburg, USA) using the in Supplementary Table 3 listed primers, Morrbid and Bim primers were analogous to Kotzin et al. (2016). Reactions were performed and analyzed using a LightCycler 480 Instrument II (Roche, Rotkreuz, Switzerland). The 2−ΔΔCt method (Livak and Schmittgen 2001) was used for the relative quantification of the measured gene expression levels. To ensure comparability among the analysis, the housekeeping gene HPRT was included as internal control and the cell lines HL-60/KG-1 alpha were included as calibrator. Serum IL-5 was measured at baseline using a human IL-5 ELISA Kit (Biorbyt, Cambridge, UK). Reactions were performed and analyzed in a GloMax Discover Microplate Reader (Promega, Fitchburg, USA).
Statistical analysis
The performed statistical tests are indicated in the figure legends. p values have not been adjusted for multiple testing, are two-sided and with a significance level of 0.05. Statistical analyses were performed using GraphPad Prism 7.0e (GraphPad, San Diego, USA).