Functional Genomics Analysis of Dendritic Cell Development

Dendritic cells (DC) represent the most potent antigen presenting cells of the organism and are of high clinical interest for the development of cell based anti-tumor immunotherapy strategies. We recently demonstrated for the first time that human DC can be generated at high efficiency in clinically approved defined serum-free cultures of purified CD34+ hematopoietic progenitor cells. DC develop from CD34+ cells along a myeloid differentiation pathway involving myeloid-committed progenitor cells (MPC) which require a combination of synergistically acting positive regulatory hematopoietic growth factors (including GM-CSF, IL-3 SCF, FL and TPO) for vigorous proliferation and expansion. These MPC subsequently require additional cytokine stimuli such as TNFalpha plus TGF-beta1 or calcium ionophore (CaI) stimulation for lineage differentiation into DC. CaI additionally induces DC differentiation and maturation from malignant MPC from acute myeloblastic leukemia cells (AML) which have lost normal cytokine responsiveness. Despite substantial knowledge on the cellular and functional characteristics of DC and their precursors, very little is known about the molecular and genetic mechanisms which control DC development and differentiation. In this proposal we will use the powerful tools of retroviral cDNA library screening for identifying genes in living MPC which regulate DC differentiation using two screening assay systems. In a the first system we will identify anti- apoptotic and/or pro-proliferative genes of appropriate cDNA libraries which confer resistance to cytokine withdrawal of normal MPC generated from CD34+ cells. In the second assay, we will screen for cDNAs which induce DC maturation of normal MPC or HL60 AML model cells using a cDNA library prepared from CaI induced mature DC. Retroviral cDNA inserts, of selected cells in these assays will be re-screened, functionally verified and subsequently analyzed in detail using secondary assay systems. This novel functional genomics approach will enhance our basic understanding of DC development and may lead to the identification of molecular targets for modulating DC function in vivo. Influence of the proposed work on the development of the field Using the here described genetic screening systems, we expect to identify molecules which play critical roles in the physiology and pathophysiology of immature normal and malignant myeloid cells and their development into mature DCs. The identification of these molecules will be of relevance for characterization of moleular pathways regulating myelopoisis and DC devlopment, about which presently only little is known. This information will complement already available detailed data about differentiation processes at the cellular level. Identified cDNAs by our screening approaches can be immediately tested in previously described differentiation models of CD34+ cells using gene transfer experiments, therefore the role of these molecules during distinct stages of hematopoietic development can be studied in detail. Apart from enhancing our basic understanding of myelopoietic and DC devlopment at a molecular level, we expect our functional screening approach will provide information on molecular targets for future studies aimed at modulating DC maturation in vivo.