Cd14 monocytes

Human tumor cell lines U937, THP-1, Raji and SK-BR-3 were obtained from the American Type Culture Collection and cultured as recommended by the vendor. Human PBMCs, CD14+ monocytes, neutrophils and dendritic cells were purchased from AllCells.

Two types of human monocyte-derived macrophages (h-Mo-Mφs) were generated as described in Supplementary Figure S1A. CD14⁺ monocytes (AllCells, Alameda, CA, USA) were cultured (37°C, 5% CO₂) in RPMI1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml human granulocyte macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, Bergisch Gladbach, Germany) or 100 ng/ml human macrophage colony-stimulating factor (M-CSF; R&D Systems) for 7 days. The M-CSF-treated cells were further cultured in RPMI1640 supplemented with 10% FBS, 100 ng/ml human M-CSF, and 20 ng/ml human interleukin (IL)-4 (R&D Systems) for 2 days. Human monocyte-derived DCs (h-Mo-DCs) were obtained as shown in Supplementary Figure S2B. For h-Mo-DC generation, CD14⁺ monocytes were cultured in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 100 ng/ml human GM-CSF and 100 ng/ml human IL-4. Cells were incubated at 37°C in an atmosphere containing 5% CO₂ for 6 days, with medium changes on days 2 and 3. Generated cells were harvested on day 6 and reseeded in fresh medium supplemented with 100 ng/ml GM-CSF and 100 ng/ml IL-4. To generate mature DCs, cells were further supplemented with 10 μg/ml CD40 agonist antibody (patent, WO/2002/088186). The characteristics of monocyte-derived cells were verified by flow cytometry (FCM).

Pacritinib was provided by CTI BioPharma Corp. (Seattle, WA, USA). Ruxolitinib, fedratinib, and momelotinib were obtained from Selleckchem (Houston, TX, USA). Primary human endothelial cells (HuVEC), neonatal foreskin fibroblasts (HDFn), bronchial epithelial cells (BE), coronary arterial smooth muscle cells (CASMC), and keratinocytes (HEK) were obtained from Lonza, Lifeline Cell Technologies and Cell Applications. Peripheral blood mononuclear cells (PBMCs) and positively selected primary normal human CD19+ B cells, CD4+ T cells, and CD14+ monocytes were purchased (AllCells, Emeryville, CA) or were isolated in-house (LeukoPak [StemCell and Physicians Plasma Alliance]). All primary cells were obtained under protocols that were reviewed by Institutional Review Board(s) that operate in accordance with the requirement of EPA Regulation 40 CFR 26 and HHS Regulation 45 CFR 46 of the US Department of Health and Human Resources for the protection of human research subjects. Stimuli include recombinant human IFNγ, TNFα, and IL-1β, IL-4, PDGF, FGF, EGF, and TGFβ (Peprotech, SEB, and TSST [Toxin Technologies]) Histamine, LPS, (Sigma), anti-human IgM (Southern Biotechnology), and Zymosan (Invivogen). Mouse detection antibodies against human biomarkers were obtained from commercial sources, including BD BioSciences, Sigma, and R&D Systems.