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Blood, 15 April 2002, Vol. 99, No. 8, pp. 2897-2904
IMMUNOBIOLOGY
Spontaneous generation and survival of blood dendritic cells in
mononuclear cell culture without exogenous cytokines
Christopher S. K. Ho,
David Munster,
Christopher M. Pyke,
Derek N. J. Hart, and
J. Alejandro López
From the Dendritic Cell Laboratory, Mater Medical
Research Institute, Brisbane, Australia; and the University of
Queensland Department of Surgery, Mater Misericordiae Hospital,
Brisbane, Australia.
Studies on purified blood dendritic cells (DCs) are hampered by
poor viability in tissue culture. We, therefore, attempted to study
some of the interactions/relationships between DCs and other blood
cells by culturing unseparated peripheral blood mononuclear cell (PBMC)
preparations in vitro. Flow cytometric techniques were used to
undertake a phenotypic and functional analysis of DCs within the
cultured PBMC population. We discovered that both the
CD11c+ and CD11c CD123hi DC
subsets maintained their viability throughout the 3-day culture period,
without the addition of exogenous cytokines. This viability was
accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts
increased (for CD123hi and CD11c+ subsets)
after overnight culture of PBMCs. Single-cell lineage depletion
experiments demonstrated the rapid and spontaneous emergence of
"new" in vitro generated DCs from
CD14+/CD16+ PBMC radioresistant precursors,
additional to the preexisting ex vivo DC population. Unlike
monocyte-derived DCs, blood DCs increased dextran uptake with culture
and activation. Finally, DCs obtained after culture of PBMCs for 3 days
were as effective as freshly isolated DCs in stimulating an allogeneic
mixed leukocyte reaction.
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