Progenitor recruitment and in vitro expansion of immunostimulatory dendritic cells from human CD34+ bone marrow cells by c-kit-ligand, GM-CSF, and TNFα

Abstract

Several cytokines have been identified that support the development of dendritic celts from murine and human precursor populations, most notably GM- CSF, TNFα, and IL-4. We have been interested in human bone marrow as a source of defined CD34+ progenitors to generate large numbers of autologous dendritic cells for use as adjuvants in immune based therapy. In serum- replete conditions with c-kit-ligand, GM-CSF, and TNFα, dendritic cells constitute ~ 1015% of the myeloid progeny (equivalent to ~ 1.7 x 106 dendritic cells per single ml of starting bone marrow); and they develop together with granulocytic intermediates and monocytes in the same cultures. CD 14- dendritic cells share expression of class II MHC and costimulatory ligands with CDI4+ monocyte progeny, but only the CDI4- HLA-DR+ dendritic cells are highly stimulatory of resting unprimed T cells. We have further identified a novel colony that develops in the presence of GM-CSF and TNFα alongside typical CFU-GM, which is comprised of dendritic cells mixed with ≤ 15% monocytes (CFU-DC/mono). c-kit-ligand recruits and expands early progenitors responsive to the dendritic cell-differentiating effects of GM- CSF and TNFα, effecting a 100- to 1000-fold greater expansion of CFU- DC/mono by 14d and 21d respectively than does the combination of GM-CSF and TNFα without c-kit-ligand. Conclusive proof that dendritic cells have been generated under these in vitro conditions has been founded on the morphologic, phenotypic, and functional criteria that are critical to their identification as potent accessories for primary T cell responses, and their distinction in particular from monocytes.