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In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)-based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1 + CD14 – MHC-II + CD172a –/ lo CD1 – CD163 – DEC205 + CD11R3 lo CD11R1 + CD33 + CD80/86 + . They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4 + CD8 + T cells, but were deficient in phagocyting inactivated Staphylococcus aureus ( S. aureus ). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1 + CD14 – MHC-II + C D172a + CD1 + CD163 –/ lo DEC205 lo CD11R3 + CD11R1 + CD33 + CD80/86 + ; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4 + CD8 – /CD4 + CD8 + T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14 + population was identified with the phenotype CADM1 + CD14 + MHC-II + CD172a + CD1 + CD163 + DEC205 – CD11R3 + CD11R1 + CD33 –/ lo CD80/86 + . They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14 + population was efficient ...

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