Generation of murine dendritic cells from flt3-ligand–supplemented bone marrow cultures

Abstract

Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

ISCOMATRIX vaccines mediate CD8+ T‐cell cross‐priming by a MyD88‐dependent signaling pathway

“…A characteristic feature of TLR‐induced DC activation is increased cell surface expression of T‐cell co‐stimulatory molecules and MHC class II 13 , 32 . To establish whether ISCOMATRIX adjuvant directly activated DCs, we generated DCs in vitro using the FMS‐like tyrosine kinase 3 (Flt3) ligand culture system 33 , 34 . Compared with TLR4 (lipopolysaccharide, LPS) or TLR9 (CpG) stimulation, ISCOMATRIX adjuvant failed to induce CD40, CD69, CD80 or MHC class II expression (Figure 5a and data not shown).…”

Murine dendritic cell development: Difficulties associated with subset analysis

“…Maturing LC en route to the lymph node up‐regulate CD8α expression 95 . The same is also true for BM‐derived DC activated with LPS or IFN‐α 96 . Recently it was shown that a proportion of purified splenic CD8α − DC can up‐regulate CD8α, CD205 and CD24 following intravenous injection into mice 97 .…”

CpG-DNA aided cross-presentation of soluble antigens by dendritic cells

“…Flt3‐ligand supplemented bone marrow cell cultures were generated as described with slight modification 37. Briefly, bone marrow cells were flushed out of femurs and tibiae,centrifuged and red blood cells were lysed for 2 min with tris‐ammonium chloride at 37°C.…”