Lipid bodies organelles and the cross-presentation of cell-associated antigens by MHC class I in phagocytic dendritic cells

Lead Research Organisation: King's College London
Department Name: Immunology Infection and Inflam Diseases

Abstract

Dendritic cells (DCs) are a specific cell type of the immune system. They act as sentinels able to warn T cells to get activated and to proliferate in case of danger. Once danger has gone, some T cell specific for the antigen keep on patrolling and they are termed as memory T cells. In case of antigen reencounter, they have the interesting ability to respond faster and better. Practically, this process of memorization is the basis of vaccination during which the immune system acquire the ability to respond faster and better to a given pathogen.
We are studying the beginning of the process which is termed as "antigen cross-presentation to T cells". A specific population of DCs activates a family of T cells called CD8+ T cells that have the ability of kill antigen-bearing cells like cells infected by virus during viral infection, for instance. Antigen cross-presentation by DCs, in this case, takes place because DCs can eat (phagocytose) dead cells that carry antigens. After their meal, DCs digest dead cells and then sample antigens (peptide molecules) out of the dead cell body to present them on display molecules (called MHC class I) at their surface. CD8+ T cells see the association of peptides with MHC molecules at the surface of the DCs.
The focus of our research is : what is happening during the digestion ? how the small antigen get extracted from the dead cell body that had been eaten by the DC ?
We had already found an organelle dedicated to store fat (lipid bodies) that regulates antigen cross-presentation. We want to understand better how it work. This research grant intend to test the function of some genes that enable to enable cells to eat a little pieces of themselves to produce energy quickly in case of stress. This process is called autophagy and also target these lipid bodies. We wonder is autophagy may not regulate, perhaps through lipid bodies regulation, the digestion of dead cell bodies and the production of the peptide antigen-MHC class I complexes. To test this hypothesis, we are going to develop some mutant mice in which autophagy genes or some regulator of autophagy are inactivated into DCs.
We will use these mice to analyse what autophagy is doing during the phagocytosis of dead cells and to determine if autophagy is important for the cross-presentation of antigens to CD8+ T cells.

Technical Summary

This proposal intend to analyze the contribution of Atg7 and of the Rab32 small GTPase to the cross-presentation of cellular antigens by phagocytic MHC class I in CD8+/CD103+ dendritic cells (DCs). The following mouse models for DC-specific gene inactivation will be generated :
- Atg7lox/lox mice (M. Komatsu, Tokyo) X CD11c-cre DC-specific deleter strain (Jackson lab).
- Rab32lox/lox (KOMP consortium) X CD11c-cre DC-specific deleter.
We will decipher the function of autophagy during the phagocytic uptake of dead cells by DCs using cutting-edge microscopic approaches on Flt3L-derived bone marrow-derived DCs. Auto-phagosomes (monitored by LC3/Atg8-GFP and PtdIns(3)P-specific FYVE domain-GFP reporters), synthesis of lipid bodies (LBs, labelled with specific probes) and phagosome maturation will be monitored in various genetic settings. Dead fibroblast carrying model antigen (OVA) will be used as a phagocytic substrate for cross-presentation.
Over-expression experiments of Rab32 (and activity-deficient mutants) into WT or autophagy-deficient DCs (CD11c-crexAtg7lox/lox) or LBs-deficient DCs (Adrp/Plin2-/-) will be performed to test the coordination of various genes and its impact on cross-presentation DCs.
Finally, we will develop an in vivo model to test the function of Atg7 and Rab32 on cross-presentation by DCs in a physiological settings : presentation incompetent mouse recipients expressing a transgenic model antigen in all tissues (Ubi-OVA x H-2Kbm1/bm1) will be irradiated and reconstituted with H-2Kb/b bone marrow from WT or CD11c-crexAtg7lox/lox or CD11c-crexRab32lox/lox. After full hematopoietic reconstitution, constitutive cross-presentation of tissue model antigen (OVA) by donor bone-marrow derived DCs (of various genotypes) will be assessed using adoptive transfer of CFSE-labelled OVA-specific OT-I transgenic CD8+ T cells. This experiment should definitively address the role of Atg7 and Rab32 autophagy-promoting genes in cross-presentation by DCs.

Planned Impact

Understanding cross-presentation by dendritic cells and its regulation by lipid storage is of clinical relevance in various fields of medicine in which cross-presentation controls clinically-important CD8+ T cell responses.
Specifically, this research proposal may participate to define new therapeutic targets. These targets might be manipulated in order to improve (in the case of vaccine, immunotherapies) or decrease (auto-immunity, graft rejection) cross-presentation efficiency.
Also, more directly, understanding how dendritic cells handle dead cell corpses to produce MHC class I peptides may fuel the development of rationale immunization strategies based on dendritic cell targeting.
Besides, the research on metabolic dysfunction and obesity may also take advantage of a better knowledge of the cell biology of lipid droplets. For instance, inhibitors of acyl-transferases involved in lipid bodies biosynthesis have been proposed as anti-obesity drugs. Other intervention on lipid bodies transport and integration in the autophagy pathway may also help to reduce intracellular lipid stores.
For all these reasons, this research is likely to generate commercially exploitable intellectual property and research tools as well. We will make use of available structures for industrial outreach at KCL to help identify opportunities of productive interactions with industrial partners. CASE awards may serve to fund research personal at the interface between our lab and industrial partners.
 
Description Arthritis Research UK
Amount £839,000 (GBP)
Organisation Versus Arthritis 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2014 
End 01/2019
 
Description King's Health Partners Research and Development Challenge Fund
Amount £90,000 (GBP)
Organisation King’s Health Partners 
Department King's Health Partners Research and Development Challenge Fund
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
End 11/2015
 
Title ESTABLISHMENT OF DC KNOCK-OUT LINES 
Description In our project we are developing tools to efficiently investigate new mechanisms involved in cross-presentation. In orther to achieve this goal we are using a new murine DC line, named MutuDC, developed at the University of Lausanne (Fuertes Marraco et al., 2012) to generate stable knock-out cells of several genes which represent potential targerts in the regulation of cross-presentation. The production of knock-out Mutu cells is based in the recently described CRISPR (clustered regularly interspaced short palindromic repeats) technology to perform efficient genome engineering in eukaryotic cells (Shalem et al, 2014; Ann Ran et al., 2013) 
Type Of Material Cell line 
Provided To Others? No  
Impact Currently there is any cellular model to perform genetic studies in DC immortalized cell lines. The only methods available rely on procedures aimed to generate transient or satble knockdown cells throgh siRNA transfection or shRNA transduction. These methods are limited by the knockdown efficiency, the presence of off-target effects difficult to assess, by technical difficulties (for instance the need to employ inactive retrovirus or lentivirus to transduce the cells) and by the difficulty to generate an stable, homogeous cell line. So far, the only alternative method to overcome these limitations is the generation of knock-out mice. By employing the CRISPR technology on a DC immortalized cell line, we are in the process to generate knockout cells which can be used as a model to study key mechisms in the biology of DC. The use of this technology can also provide an efficient and economic cellular model to replace the use of mouse models. 
 
Title Establishement of in vivo assay for the metabolic status of phagocytic dendritic cells 
Description Using a FACS based assay in mice receiving frluorescent beas, we had been able to measure the cellular ROS and mitochondrial mass in cells having phagocytosed particles in vivo. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact This technique will be used to further delineate the phenotype of Rab32 ko mice. 
 
Title GENERATION OF A RAB32 KNOCKOUT MOUSE 
Description We are developing a Rab32 knockout mouse model. Transgenic heterozygous mouse embryos were obtained from the knockout mouse project (KOMP) repository. These transgenic mice carry a lacZ reporter gene and a neomycin resistance gene inserted between exons 1 and 2 of the Rab32 gene. The lacZ reporter gene is initiated by a splicing acceptor sequence and terminates with a stop codon, therefore homozygous mice carrying the transgene are Rab32 knockout mice. This model of knockout is referred as tm1a(KOMP)Wts. Moreover this straight knockout can be further converted into a conditional knockout as the lacZ and the nemoycin resistance genes are flanked by loxP and FRT sequences. Crossing the straight knockout mice tm1a(KOMP)Wts with a Flipase transgenic mouse will delete the whole construct (loss of the lacZ and neomycin resistance genes), just leaving the loxP sites flanking the exon 2 of the gene. The result of this intermediate step is the recovery of the normal expression of the wt form of the Rab32 gene. A further crossing of these mice with a transgenic mice expressing the Cre recombinase under the control of a specific cell line promoter (like CD11c for dendritic cells) will lead to the deletion of the exon 2 of the protein in that specific cell lineage (conditional knockout). 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The generation of knockout mice for Rab32 will allow us to study the impact of Rab32 function in in vivo studies. As our project is mainly focused in understanding the molecular mechanisms regulating antigen presentation by dendritic cells, the generation of Rab32 knockout mice will be of great value to further complete the data provided by our in vitro cellular models 
URL https://www.komp.org/alleles.php#conditional-promoter-csd
 
Title GENERATION OF IMMORTALISED RAB32 KO HEMATOPOIETIC CELL LINES WITH MYELOID AND LYMPHOID POTENTIAL 
Description Currently we have generated homozygous mice carrying the tm1a (KOMP)Wts allele, but in order to minimize the use of these mice for in vitro studies related to the function of immune cells we are trying to generate immortalised bone marrow cells through the transduction of an estrogen-regulated form of the Hoxb8 oncogene following the protocol described by Redecke et al. (Nature methods, 2013). Importantly, immortalised bone marrow cells following this method can be in vitro differentiated into macrophages, granulocytes, dendritic cells, B lymphocytes and T lymphocytes that are phenotypically and functionally indistinguishable from their primary counterparts. 
Type Of Material Cell line 
Provided To Others? No  
Impact The generation of a Rab32 knockout cell line with the capacity to be in vitro and in vivo differentiated into myeloid and lymphoid cells will allow us to perform in vitro functional studies reducing the use of mice. 
 
Description Analysis of the role of Rab32 in MHC trafficking 
Organisation Curie Institute Paris (Institut Curie)
Country France 
Sector Academic/University 
PI Contribution The goal of the collaboration is to analyse if Rab32 controls MHCI and MHCII trafficking
Collaborator Contribution Granted access to MHCII-GFP mice and Ii-deficient mice
Impact in progress
Start Year 2015
 
Description Analysis of the role of the BLOC3 in crosspresentation by MHCI 
Organisation University of Aberdeen
Department Department of Archaeology
Country United Kingdom 
Sector Academic/University 
PI Contribution We are analysing the contribution of the Rab32 GTP exhange factor BLOC3 in crosspresentation by MHCI in dendritic cells
Collaborator Contribution Dr Spano granted access to bone marrow of BLOC3-deficient mice
Impact in progress
Start Year 2016
 
Description Dr Noboru Mizushima, Tokyo University 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution Analysing the role of autophagy in lipid bodies dynamics in DCs.
Collaborator Contribution Providing Atg5/lox/lox embryos and GFP-LC3 transgenic mice.
Impact This collaboration should define the molecular machinery underlying the formation and maintenance of lipid bodies organelles in DCs.
Start Year 2014
 
Description Dr. Loredana Saveanu. 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution Our group, in collaboration with the group of Dr. Loredana Saveanu (INSERM U1151/Bichat Hospital), are working together on the mechanisms by which Rab GTPases regulate corss presentation in DCs. This leads to the exchange of numerous protocols and expertise for example in the field of in vitro models of bone marrow derived DCs, lentiviral transduction, cellular imaging. In addition, we are working on the submission of parallel reviews about cross-presentation as a part of an special issue hosted in the journal frontiers in Immunology titled: "Unconventional antigen presentation: atypical cells and unusual pathways". An abstract of the review has already been accepted by the editors of the journal. Our contribution to their review will include bibliographic assessment, dessign and evaluation of the manuscript.
Collaborator Contribution This leads to the exchange of numerous protocols and expertise for example in the field of in vitro models of bone marrow derived DCs, lentiviral transduction, cellular imaging.
Impact The expected evaluation of the reviews for the publication in the journal will be in 01 Apr 2015.
Start Year 2006
 
Description University of California DAVIS 
Organisation University of California, Davis
Department UC Davis College of Biological Sciences
Country United States 
Sector Academic/University 
PI Contribution We are handling the the derivation of live Rab32-targeted mice.
Collaborator Contribution The KOMP repository provides us with embryos genetically targeted for Rab32 with a targeting construct allowing the production of conditional knock-out.
Impact The generation of live Rab32 conditional ko will serve the analysis of its role in dendritic cells and in the generation of adaptive immunity more broadly.
Start Year 2014
 
Description Laboratory visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Mini internship for GCSE student

Stimulation of scientific interest and general awareness about science
Year(s) Of Engagement Activity 2014