Macrophage csf m csf

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy individuals and patients by density gradient centrifugation with Lymphoprep (#AXS-1114547; Cosmo Bio USA, CA, United States). Monocytes isolated from PBMCs were isolated with EasySep Human Monocyte Enrichment Kit (#19058; Stemcell Technologies), or by plastic adherence as previously described.¹⁹ (link) Monocytes were differentiated into Mφ by treatment with 20 ng/mL macrophage-CSF (M-CSF) (#574806; BioLegend, CA, United States) for 5-day as reported.¹⁹ (link) Mφ were further differentiated by stimulating with 100 U/mL IFN-γ (#11725-HNAS; Sino Biological; PA, United States) and 100 ng/mL LPS (#L2630; MilliporeSigma, MA, United States). Mφ were detached from plates using Accutase Cell Detachment Solution (#AT-104; Innovative Cell Technologies, CA, United States) and Cell Scraper (#3010; Corning, NY, United States). Naive CD4 T cells and total CD4 T cells were purified using the EasySep human naive CD4 T cell and human CD4 T cell isolation kits (#19555, #17952; Stemcell Technologies, Vancouver, Canada). Cells were cultured in RPMI 1640 medium (#11875135; Thermo Fisher Scientific, MA, United States) supplemented with 10% FBS (#100–106; GeminiBio, CA, United States) and were incubated at 37°C with 5% CO₂.

All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Fetal bovine serum (FBS; Gibco, Waltham, MA), 100× l-glutamine, 100× penicillin/streptomycin HyClone (Pittsburgh, PA), and Gibco 100× antibiotic mix were obtained from Thermo Fisher Scientific (Waltham, MA). RPMI 1640, Dulbecco’s modified Eagle’s medium (DMEM), and Matrigel are from Corning (Tewksbury, MA). Mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 6 (IL-6), IL-4, macrophage CSF (M-CSF), and FMS-like tyrosine kinase 3 ligand (FLT3L) were obtained from BioLegend (San Diego, CA). OVA was obtained from Thermo Fisher Scientific. Mouse IFN-γ ELISA kit was obtained from R&D Systems (Minneapolis, MN). Mouse CD4⁺ T cell isolation kit and CD8⁺ T cell isolation kit were obtained from Miltenyi Biotec (Auburn, CA). Clodronate and control liposomes were obtained from Liposoma (Amsterdam, The Netherlands). In vivo anti-mouse CD40, anti-mouse PD-1, anti-mouse Ly6C monoclonal antibodies, and their controls (rat IgG2a) were all obtained from Bio X Cell (Lebanon, NH). KO-validated PKCδ antibody and phycoerythrin/Cy7 conjugation kit were obtained from Abcam (Cambridge, UK). Flow cytometry antibodies, compensation beads, and reagents are described in table S1 [Tonbo Biosciences Inc. (San Diego, CA), Thermo Fisher, and BioLegend].