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

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.

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.

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.