Cellular Immunology Project, Section of physiology and cell biology. NorMIC IBV imaging facility.
Immune cells rely on information conveyed by fragments of intracellular components being presented on major histocompatibility complex (MHC) molecules on the cell surface. Many pathogens are too large to be recognized directly by immune cells and must first be digested into smaller fragments (peptides) that can be presented by specialized antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages.
Cancer immunotherapy attempts to harness the power and specificity of the immune system to treat tumors. The molecular identification of cancer-specific antigens has allowed the development of antigen-specific personalized immunotherapy. Due to their properties, DCs are often called 'nature's adjuvants' and thus have become a leading vehicle for antigen delivery.
MHC class II associated invariant chain (Ii) or CD74 (Cluster of Differentiation 74) is a protein essential for proper formation and trafficking of MHC class II to the antigen loading compartment and thus efficient antigen presentation. (se Nefjes… Bakke, Nature Rev. Immunollogy, 2011) Ii induces a delay in endosomal maturation and an enlargement of early endosomes dependent on its cytoplasmic domain. This function is independent of the normal early endosomal fusion machinery comprising the small GTPase Rab5, PI3 kinase and the tethering factor EEA1.
All membrane fusion events in eukaryotic cells are mediated by SNARE (soluble NSF attachment protein receptor) proteins. Four different SNARE-domains (Qa, Qb, Qc and R) are needed to form a functional complex. We have recently performed an siRNA screen to identify SNAREs and SNARE-regulating proteins involved in Ii-induced endosome enlargement.
The aim of this master project is to characterize the specific SNARE complex in Ii-positive endosomes and its functional significance in antigen presentation. The master student will study the localization and interaction of SNARE proteins by immunofluorescence, expression of fluorescent protein-tagged SNARE proteins and confocal microscopy, depletion of target proteins, biochemistry, immunoprecipitations etc. The experimental set up is also based on antigen presenting cell lines where invariant chain is KO by CRISPR/Cas9 and cells where the different fluorescent Rabs are inserted by the same method. The project is in collaboration with a Dutch group with whom we have long experience and published together. The group in Leiden University is led by Professor Jacque Neefjes see https://ccb.lumc.nl/research/chemical-immunology-171/jacques-neefjes-155. The project is performed in collaboration and may include travelling and work the Neefjes lab in Leiden.
Methods
The project will take place in the group of Oddmund Bakke/Cinzia Progida at their common laboratory and imaging facility, NorMIC Oslo imaging platform. The student will in particular learn several state-of-the-art microscopy techniques image processing and quantitative image analysis that are very much in demand both in academic research and in companies.
The project also includes
Molecular biology techniques and cell biology, including mammalian cell culture, gene manipulations, protein expression and purification, and immunoprecipitation etc. and is particularly suited for a motivated candidate that have a wish to continue working with cellular/molecular research and imaging. The project is a basis for the further development of a cancer immunotherapy vaccine (W?lchli S et al.Eur J Immunol. 2014 Mar;44(3):774-84. doi: 10.1002/eji.201343671) and Kucera et al 2020, https://www.biorxiv.org/content/10.1101/2020.05.13.091579v1 ) and recent work on the SNARES that are not yet published.
Project background and methods in imaging by the advisors are also well described in:
- Margiotta A, Frei DM, Sendstad IH, Janssen L, Neefjes J, Bakke O. Invariant chain regulates endosomal fusion and maturation through an interaction with the SNARE Vti1b. J Cell Sci. 2020 Oct 9;133(19):jcs244624. doi: 10.1242/jcs.244624. PMID:
- Skjeldal FM, Haugen LH, Mateus D, Frei DM, R?dseth AV, Hu X, Bakke O. De novo formation of early endosomes during Rab5-to-Rab7a transition. J Cell Sci. 2021 Apr 15;134(8):jcs254185. doi: 10.1242/jcs.254185. PMID: 33737317; PMCID: PMC8106955.
- General info in our review in Nature: Neefjes J, Jongsma ML, Paul P, Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol. 2011 Nov 11;11(12):823-36. doi: 10.1038/nri3084. PMID: 22076556.
Contacts (to be found also at the 3rd floor Kristine Bonnevies hus)
Oddmund Bakke, professor em., Main Supervisor
Xian Hu, co-supervisor
Frode Skjeldal, co-supervisor
Cinzia Progida, professor co-supervisor
???????phone: +47 22855787/95851479. email: oddmund.bakke@ibv.uio.no