Periodic Reporting for period 1 - DC Metabolism (Metabolic Regulation of Conventional Dendritic Cell Development and Function)
Reporting period: 2020-07-01 to 2022-06-30
Dendritic cells (DCs) are key sentinel immune cells that alert the body to the presence of damage or infection, and arm T-lymphocytes to expand and eliminate aberrant or infected cells. Despite their importance in immunity, the mechanisms that govern their development and specific functions are not fully understood. Conventional DCs can be subdivided into type 1 (cDC1) and 2 (cDC2) subsets, which both descend from a common precursor under the shared influence of the growth factor cytokine Flt3L. The signals that differentiate cDC1 from cDC2 during development remain unknown. Apart from development, cDC1 and cDC2 also have specialized roles in immune responses. cDC1 are important for arming CD8+ T cells by processing and presenting proteins derived from cancer or infected cells through a process termed cross-presentation, whereas cDC2 help mediate immunity to parasites and extracellular bacteria. The importance of cDC1 in controlling immune responses against cancer is established by numerous studies demonstrating that a lack of cDC1 in mice impairs their rejection of tumours and responses to immune checkpoint blockade and adoptive T cell therapies. However, the precise mechanisms underlying the ability of cDC1 to cross-present are unclear. Imbedded in immune cell physiology are metabolic pathways and metabolites that not only serve to provide energy and breakdown or produce nutrients for growth and survival, but also to instruct their development and function. For example, failure to engage specific metabolic pathways has been found to impair the development of memory T cells and the activation of macrophages, another type of sentinel immune cell.
This project aims at delineating the role and importance of metabolic regulation in DC basic biology by answering two main questions: do distinct metabolic programs dictate cDC1 versus cDC2 specification, and what are the metabolic pathways or nutrients required for cDC1-dependent cross-presentation? Understanding the role metabolism plays in directing DC physiology will identify novel mechanisms of immune cell control with implications for antiviral and anticancer immunity.
The conclusions of the action are thus far: the metabolic profiles of cDC1 and cDC2 were assessed and a specific isoform of hexokinase, a protein essential to the metabolism of sugar, was found to be expressed primarily by cDC1. Preliminary data using novel reagents suggests that this isoform may be critical to cDC1 differentiation and survival.
This project aims at delineating the role and importance of metabolic regulation in DC basic biology by answering two main questions: do distinct metabolic programs dictate cDC1 versus cDC2 specification, and what are the metabolic pathways or nutrients required for cDC1-dependent cross-presentation? Understanding the role metabolism plays in directing DC physiology will identify novel mechanisms of immune cell control with implications for antiviral and anticancer immunity.
The conclusions of the action are thus far: the metabolic profiles of cDC1 and cDC2 were assessed and a specific isoform of hexokinase, a protein essential to the metabolism of sugar, was found to be expressed primarily by cDC1. Preliminary data using novel reagents suggests that this isoform may be critical to cDC1 differentiation and survival.
Using Seahorse Extracellular Flux Analysis and advanced liquid and gas chromatography coupled mass spectrometry, I was able to establish using both in vitro derived and ex vivo isolated specimens that cDC1 and cDC2 have distinct metabolic profiles and mitochondrial activities. Consistent with previous observations, I found that glucose metabolism appears to be key to DC sensing of pathogen derived signals, while the role of fatty acid metabolism in cDC1-dependent cross-presentation remains unclear. I also developed a new reagent of conditionally immortalized DC progenitor (ER-Hoxb8) cells that can be used to perform genetic loss and gain-of-function to assess questions pertaining to DC development and function.
After discovering that cDC1 express a unique isoform of the glycolytic enzyme hexokinase, I used the newly created ER-Hoxb8 cells to show that this enzyme is essential for cDC1 differentiation and critical for DC survival in vivo. CRISPR mediated knockout mice of this gene were created to facilitate further studies, however no difference was seen between these animals and wild-type controls using a variety of assays including tumour transplantation. Increased expression and activity of the other isoforms of the enzyme appeared to compensate for the loss of the cDC1-specific isoform, which may explain the discrepancy observed between the results I obtained with ER-Hoxb8 cells and germline deleted knockout mice. Therefore, a novel mouse is currently being produced that will allow me to conditionally delete this gene in a cell-type and time-specific manner.
This work is ongoing and will be compiled alongside new developing research into a publication as a scientific article and has been presented at various scientific workshops and conferences including the Crick Immunology Interest Group and Immunobiology Lab Retreats. All the datasets and reagents produced including the ER-Hoxb8 cells will be made available to other researchers upon request. No patents have been filed to exploit the results from the action at this time.
After discovering that cDC1 express a unique isoform of the glycolytic enzyme hexokinase, I used the newly created ER-Hoxb8 cells to show that this enzyme is essential for cDC1 differentiation and critical for DC survival in vivo. CRISPR mediated knockout mice of this gene were created to facilitate further studies, however no difference was seen between these animals and wild-type controls using a variety of assays including tumour transplantation. Increased expression and activity of the other isoforms of the enzyme appeared to compensate for the loss of the cDC1-specific isoform, which may explain the discrepancy observed between the results I obtained with ER-Hoxb8 cells and germline deleted knockout mice. Therefore, a novel mouse is currently being produced that will allow me to conditionally delete this gene in a cell-type and time-specific manner.
This work is ongoing and will be compiled alongside new developing research into a publication as a scientific article and has been presented at various scientific workshops and conferences including the Crick Immunology Interest Group and Immunobiology Lab Retreats. All the datasets and reagents produced including the ER-Hoxb8 cells will be made available to other researchers upon request. No patents have been filed to exploit the results from the action at this time.
Firstly, work from this project proposal allowed me to acquire skills and expertise in DC biology, antiviral immunity, and cancer. The skills and expertise I gained during the last two years of work as a postdoc within the scientific environment at the Francis Crick Institute are reflected in several projects published in highly renowned journals (Cell, Science Immunology, Nature Immunology). With my colleague Enzo Poirier, I helped investigate the role of antiviral RNA interference (Poirier, Buck et al., Science 2021). During the COVID-19 pandemic, I worked with other Crick scientists to improve SARS-CoV-2 diagnostic testing (Buck, Poirier et al., Wellcome Open Research 2021 and publications in the Lancet, Nature Biotechnology).
Secondly, my postdoctoral work on DCs and metabolism allowed me to initiate a follow up project in which I am investigating how innate immune receptors expressed by DCs interface with metabolism to control their activation and ability to arm T cells. This project is being prepared for publication.
Thirdly and most importantly, the funding of my postdoctoral work in Caetano Reis e Sousa’s lab allowed me to develop my own area of research interest within the field of immunometabolism. The unique combination of expertise, tools and techniques I acquired during my research as a Marie Skłodowska-Curie fellow ideally positioned me to reach the next level of professional maturity as an independent group leader.
The discovery that cDC1 and cDC2 display specific metabolic programs and enzymes that may direct their specification and survival advances our understanding of DC biology. Modern therapies against cancer utilise the power of the immune system to directly attack and eliminate tumours, highlighted by the recent Nobel Prizes awarded to James Allison and Tasuku Honjo for their pioneering work on cancer immunotherapy. Harnessing metabolism to enhance DC specification and function may yield new or improved therapies.
Secondly, my postdoctoral work on DCs and metabolism allowed me to initiate a follow up project in which I am investigating how innate immune receptors expressed by DCs interface with metabolism to control their activation and ability to arm T cells. This project is being prepared for publication.
Thirdly and most importantly, the funding of my postdoctoral work in Caetano Reis e Sousa’s lab allowed me to develop my own area of research interest within the field of immunometabolism. The unique combination of expertise, tools and techniques I acquired during my research as a Marie Skłodowska-Curie fellow ideally positioned me to reach the next level of professional maturity as an independent group leader.
The discovery that cDC1 and cDC2 display specific metabolic programs and enzymes that may direct their specification and survival advances our understanding of DC biology. Modern therapies against cancer utilise the power of the immune system to directly attack and eliminate tumours, highlighted by the recent Nobel Prizes awarded to James Allison and Tasuku Honjo for their pioneering work on cancer immunotherapy. Harnessing metabolism to enhance DC specification and function may yield new or improved therapies.