Description of the thesis topic:
Tuberculosis is a communicable disease that remains a major cause of ill health, one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent (ranking above HIV/AIDS). According to World Health Organization, 1.4 million people died of tuberculosis in 2019 (1).
There is therefore an urgent need to develop new vaccine strategies that will allow the development of a tuberculosis vaccine that is more effective than BCG. Within the framework of international programs supported by the European Union (TBVAC2020) and the Bill & Melinda Gates Foundation, we have evaluated for several years a promising strategy that consists in using Mycobacterium tuberculosis antigens of lipidic nature in subunit vaccine formulations (2-5). These antigens are presented to T lymphocytes by the CD1 family proteins (in particular, the CD1b isoform) on the surface of antigen presenting cells (APCs). The major cellular steps in the presentation of lipid antigens by CD1 are known. However, the precise molecular mechanisms and actors involved have yet to be defined.
This thesis project aims to identify key actors and mechanisms of lipid antigen presentation by CD1b, focusing on the most poorly understood aspects of this process, including the identity of intracellular proteins required for antigen capture, trafficking to lysosomes, processing into epitopes, and loading onto CD1b. The detailed analysis of the protein composition of APC lysosomes has allowed us to identify new candidate proteins potentially involved in lipid antigen presentation such as hydrolases. Here, we will specifically inactivate selected genes by CRISPR/Cas9 technology in the APC cell lines K562-CD1b or THP1-CD1b models (6). The role of the targeted genes will be evaluated at different steps of lipid antigen presentation (capture, traffic, processing, loading) using a series of assays:
*to determine if the antigen is correctly taken up by CD1b and presented on the surface of the APC, the cells will be i) pulsed with antigenic lipids (Ac2SGL) and their capacity to activate the specific T clone will be analyzed or ii) labeled with the antibody dAbk11, which specifically recognizes the CD1b-Ac2SGL complex on the surface of the APC (7).
*in absence of T cell activation or antibody recognition, the lipid structure presented by CD1b on the surface of the APC will be investigated, using an innovative strategy we previously developed to isolate the CD1b: lipid complexes. This strategy is based on the use of APC expressing a cleavable form of the CD1b protein. Following defined conditions of cleavage and purification of the complexes, the lipids will be extracted from cleaved CD1b and analyzed by HPLC/MS.
*finally, the fate of the antigen (fluorescent synthetic form to be produced) will be analyzed by confocal microscopy from its uptake by the cells to its location in lysosomes, and its loading on CD1b (co-location by high resolution microscopy). The function of selected target proteins will be studied in vitro (cargo protein, lipid transfer protein, ...) using dedicated tests (8-10).
2. H. de la Salle et al., Assistance of microbial glycolipid antigen processing by CD1e. Science 310, 1321 (Nov 25, 2005).
3. M. Gilleron et al., Lysosomal Lipases PLRP2 and LPLA2 Process Mycobacterial Multi-acylated Lipids and Generate T Cell Stimulatory Antigens. Cell chemical biology 23, 1147 (Sep 22, 2016).
4. M. Gilleron et al., Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis. The Journal of experimental medicine 199, 649 (Mar 1, 2004).
5. G. Larrouy-Maumus et al., Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine 35, 1395 (Mar 7, 2017).
6. S. Chandra et al., Mrp1 is involved in lipid presentation and iNKT cell activation by Streptococcus pneumoniae. Nature communications 9, 4279 (Oct 15, 2018).
7. F. Camacho et al., Selection of phage-displayed human antibody fragments specific for CD1b presenting the Mycobacterium tuberculosis glycolipid Ac2SGL. International journal of mycobacteriology 5, 120 (Jun, 2016).
8. L. Blanc et al., Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids. Proceedings of the National Academy of Sciences of the United States of America 114, 11205 (Oct 17, 2017).
9. D. Cala-De Paepe et al., Deciphering the role of CD1e protein in mycobacterial phosphatidyl-myo-inositol mannosides (PIM) processing for presentation by CD1b to T lymphocytes. The Journal of biological chemistry 287, 31494 (Sep 7, 2012).
10. L. F. Garcia-Alles et al., Crystal structure of human CD1e reveals a groove suited for lipid-exchange processes. Proceedings of the National Academy of Sciences of the United States of America 108, 13230 (Aug 9, 2011).
Collaborative CNRS thesis with societal impact: the proposed project will be carried out in its entirety in the "Tuberculosis and Infection Biology" department of the Institute of Pharmacology and Structural Biology (IPBS, CNRS unit, Toulouse, France), in the team "Immunomodulation by lipids and mycobacterial glycoconjugates" led by Dr J. Nigou and in close interaction with the team "Immune detection and pathogen elimination" of E. Meunier.
How to apply:
All details are on CNRS website