Transcription factors in human DC subset differentiation

Recent progress in our understanding of the mammalian immune system led to the identification of three major sub-lineages of dendritic cells (DC). These DC sublineages (subsets: Langerhans cells, LCs; plasmacytoid DCs, pDCs; interstitial DCs, intDCs) develop via independent precursor pathways from hematopoietic stem cells in vitro. In vivo they can be found in specific anatomic compartments, and seem to possess highly specialized roles in the regulation of anti-microbial/viral immune responses. Furthermore, they might be differentially involved in the induction and maintenance of immunologic tolerance and in the pathogenesis of autoimmune diseases, allergies and cancer. Recent results showed that all known members of the DC system can develop efficiently from myeloid- committed progenitor or precursor cells in response to microenvironmental signals. These differentiation processes can be duplicated in vitro by using cultures of human CD34 + progenitor cells and peripheral blood monocytes. The underlying transcriptional processes leading to DC subset specification of myeloid progenitors and monocytes are likely similar in vitro and in vivo, and specific interference with these processes might allow to therapeutically influence immune responses. In this grant we seek to characterize the downstream transcriptional processes underlying human DC subset specification from progenitor cells and monocytes. To study this complex regulation, we will use novel powerful techniques for regulated gene induction/inhibition in phenotypically defined primary cell populations in combination with advanced flow cytometry and molecular analyses. We will: Specific Aim 1: perform a hierarchical analysis of human DC progenitors. We will functionally identify and molecularly analyze progenitors for all three DC sublineages. Furthermore, we will optimize pDC generation cultures. Specific Aim 2: Analyze candidate regulatory TFs in pDC development from common DC progenitors. We will functionally analyze the role of TFs in the subset specification of common DC progenitors, and in pDC activation. Specific Aim 3: study regulatory genes in myeloid-related conventional DCs (LCs and intDCs). We will analyze candidate TFs in monocyte-derived LC versus intDC differentiation, perform genetic validation experiments in monocytes and will establish cell line models for studying transcriptional networks in DC differentiation.

Dendritic cells (DCs) represent important cells of the immune system. They occur in most organs and tissues of the human body and are regarded as the sentinels of the immune system. Their main functions are to recognize microbial pathogens such as bacteria, viruses and fungi and to induce appropriate immune responses. DC-mediated immune responses include direct inactivation of pathogens (innate immunity) as well as the induction of antigen- specific T cell and B cell mediated responses (adaptive immunity). For activating T helper cells, DCs possess a high capacity to take up, process and present antigens in the context of additional molecules (DCs are "professonal antigen presenting cells"). Moreover, they possess a high migratory capacity, allowing them to travel from their peripheral sites to lymphoid tissues where T cell activation occurs. Additionally, they produce molecules that co- activate T cells. Since body surfaces such as skin and gut are densely populated by commensal bacteria, DCs have to discriminate between harmless and harmful microbes. To meet all these functions, at least three DC subsets have evolved. Human stratified epithelia (epidermis and mucosae) contain one specific DC subtype known as a Langerhans-type DC (LC). LCs reside in these peripheral sites for long periods of time and form dense netoworks which are highly conserved during vertebrate evolution. These cells are among all immune cells most immediately exposed to environmental substances and pathogens. LCs differ substantially from other DC subsets found in interstitial tissues or neo-recruited to inflammatory lesions. All these DC subsets can be generated in vitro from circulating monocytes. In principle, hematopoietic lineage decisions are regulated by a complex interplay of synergistic and antagonistic transcription factors. We rationalized in this project that very little is known on the identity of transcription factors during DC subset differentiation despite the fact that DCs are of substantial medical interest (e.g autoimmune/inflammatory diseases, cancer and immunodeficiency syndroms). Therefore, we aimed to identify the transcriptional mechanisms underlying human DC subset differentiation. Several transcriptional regulators were studied in this project. For instance, we identified two transcription factors involved in the reciprocal control of LC versus inflammatory DC development. Specifically, the vitamin D receptor (VDR) induces LC differentiation, whereas GATA-1 represses VDR for alternatively inducing iinflammatory DC subset differentiation. Furthermore, we identified genetic circuitries of potential importance for LC function. The aryl hydrocarbon receptor (AhR), also known as "dioxin receptor" controls LC differentiation, and microRNA (miR)- 146a seems to help allow LCs to "ignore" harmless commensal bacteria at body surfaces. We found that these two molecules AhR and miR-146a interact with a third factor, PU.1 for exerting their function in LCs.