This project aims to replace mouse use in dendritic cell research by generating an embryonic stem cell derived in vitro model.
Dendritic cells (DCs) are antigen presenting cells which act as ‘pacemakers’ in both adaptive and cell mediated immunity. Given their roles in immune responses and the maintenance of immune tolerance, investment in mouse genetics to support DC research has been significant. There are typically three approaches for studying DCs; i) extraction and purification of DCs from lymphoid organs, with around 2 x 106 cells obtained per mouse, therefore necessitating the use of large numbers of animals to provide cells for in vitro studies; ii) in vitro expansion of bone marrow haematopoetic progenitors which again uses large numbers of mice to extract bone marrow from, and iii) in vivo expansion with up to ten days administration of the growth factor Flt3L and subsequent culling of mice and purification of DCs from lymphoid organs. An in vitro model with DCs generated from mouse embryonic stem (ES) cells could provide an opportunity to replace and reduce the use of mice particularly in functional genomic
Research details and methods
DCs arise from haematopoietic progenitors expressing Flt3R/Flk2 - a lineage distinct from other leukocytes. Haematopoietic progenitors are generated spontaneously during the differentiation of ES cells into embryoid bodies. Lentivirus vectors will be used to conditionally overexpress various transcription factors in ES cells in order to evaluate their ability to drive the expansion of Flt3/Flk2+ progenitors. DCs will subsequently be terminally differentiated in growth factor dependent cultures using FLt3L. Phenotypic and functional validation ES-generated DCs will be undertaken using a range of molecular markers and assays.
Dendritic cells (DCs) are a major cell type of the immune system, coordinating the development of immune responses or the maintenance of immune tolerance. DCs arise from Flt3R/Flk2+ haematopoietic progenitors and differentiate to control lymphocyte activation by presenting antigens to CD4+ and CD8+ T lymphocytes during immune responses. For this reason, understanding DC function is of paramount importance in order to maximize protective immune response (vaccination, immunotherapy) and minimize harmful immune responses (autoimmunity). Genes expressed by DCs have been widely characterized in both mouse and human DC subsets. These studies stimulate the production of numerous genetically modified (GM) mice strains for the functional analysis of these genes. However, this type of research requires the use of many mice given the rarity of the DCs in the mouse immune system. DC expansion using Flt3L in vitro (on bone marrow cultures) or in vivo (after injection) still requires a lot of mice.
In order to reduce animal experimentation in DC research, this proposal intends to implement a new in vitro culture system to generate DCs out of murine ES cells and their genetically modified derivatives. The strategy underlying this proposal is to take advantage of genetic engineering to conditionally overexpress transcription factors expanding haematopoietic progenitors generated during the spontaneous differentiation of ES cells into embryoid bodies. The approach is to achieve conditional immortalization of progenitors to support quantitative, growth-factor independent expansion while conserving Flt3/Flk2 expression. Various transcription factors (including homeobox genes) will be evaluated in their ability to drive the expansion of Flt3/Flk2+ progenitors. Practically, we will use poly-cistronic lentiviral vectors driving regulated expression of these transcription factors upon doxycycline activation of rtTA transactivator-dependent transcription. Then, by relieving the expression of these homeobox transcription factors, DCs will be terminally differentiated in growth factor dependent cultures using FLt3L as an agonist for the Flt3/Flk2 signaling pathway. This method will be suitable to study a wide variety of genetic modification generated at the level of ES cells: gene inactivation (homozygous targeting by homologous recombination using adapted selection processes or stepwise removal of selection cassette), gene silencing (achieved by lentiviral transduction), gene overexpression, fluorescently tagged gene products. Practically, this culture system will be applied to various genes studied in the lab thus generating a major replacement of mouse experiment and breeding (hundreds of mice generated from more than 20 breedings of the GM mice that would need to be generated otherwise). If adopted by the scientific community, this method may have a broad impact on DC studies and may even stimulate developments in the study of other haematopoietic cell types. The recently opened Guermonprez lab belongs to the King's College London Guy's Hospital campus conveniently located in central London. The lab is located within the multidisciplinary Centre for Molecular and Cellular Biology of Inflammation headed by Professor Geissmann and belongs to the Division of Immunology, Infectious and Inflammatory Diseases headed by Professor Hayday. The PhD candidate will have access to a highly diverse and prestigious scientific environment at the crossroads of cell biology, immunology and inflammation research as well as high-end and state-of-the art facilities providing a stimulating work environment to achieve innovative and productive research.
Anselmi G et al. (2019). Engineered niches support the development of human dendritic cells in humanized mice. Nature Communications 11:2054. doi: 10.1101/835223
Guermonprez et al. (2019). Chapter One - Origin and development of classical dendritic cells. International Review of Cell and Molecular Biology 349:1-54. doi: 10.1016/bs.ircmb.2019.08.002
Clarke F et al. (2018). The protein tyrosine phosphatase PTPN22 negatively regulates presentation of immune complex derived antigens. Scientific Reports 8:12692. doi: 10.1038/s41598-018-31179-x
Clarke F et al. (2017). Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells. PLoS One 12(10):e0186625. doi: 10.1371/journal.pone.0186625
Adiko AC et al. (2015). Intracellular Transport Routes for MHC I and Their Relevance for Antigen Cross-Presentation. Frontiers in immunology 6:335. doi: 10.3389/fimmu.2015.00335