Anti cd5

PBMCs were isolated from buffy coats or apheresis from healthy donors (Sanquin, Nijmegen, The Netherlands) using Lymphoprep (STEMCELL Technologies). Total CD141⁺ CLEC9A⁺ DCs (cDC1s) were MACS isolated from PBMCs using the human CD141 (BDCA-3) MicroBead kit (Miltenyi Biotec), followed by isolation of total CD1c⁺ DCs (cDC2s) using the human CD1c (BDCA-1)⁺ dendritic cell isolation kit (Miltenyi Biotec). A purity check of the subsets after isolation was performed by additional FACS staining and measured flow cytometry (BD FACSCanto II). The cDC1s were stained with anti-Clec9a (SONY Biotechnology) and anti-CD141 antibodies (BioLegend), cDC2s were stained with anti-CD11c (BD Biosciences) and anti-CD1c antibodies (Miltenyi Biotec). Further cDC2 subset isolation was performed by FACS sorting (BD FACS Aria II SORP) using anti-CD11c (BC Biosciences), anti-CD1c (Miltenyi Biotec), anti-CD5 (BioLegend), anti-CD163 (eBioscience), and anti-CD14 (BD Biosciences) antibodies. The four cDC2 subsets were gated according to the following markers: P1 (CD11c⁺ CD1c⁺ CD5⁺), P2 (CD11c⁺ CD1c⁺ CD5⁻ CD163⁻), P3 (CD11c⁺ CD1c⁺ CD5⁻ CD163⁺ CD14⁻), and P4 (CD11c⁺ CD1c⁺ CD5⁻ CD163⁺ CD14⁺). Cells were cultured in X-VIVO 15 serum-free hematopoietic cell medium (Lonza) with an addition of 10% fetal bovine serum (Gibco), and 40 U/mL human granulocyte-macrophage colony-stimulating factor (hGM-CSF) (Immunotools).