Microscopy-based studies of CD1d and MR1 assisted T-cell activation
Supervisors: Prof Christian Eggeling and Prof Vincenzo Cerundolo
T-cells play a central role in cell-mediated immunity. Their immune-modulatory properties are activated by the binding of T-cell receptors (TCR) to antigen complexes on the membrane surface of accessory or antigen-presenting cells (APC). iNKT cells are an exceptional sub-population of T-cells, since their activation is restricted to the lipid-antigen presenting glycoprotein agonist CD1d. Paradoxically, effector functions of iNKT are varied and they can lead to suppressed or enhanced immune responses. Other antigen-presenting molecules include MHC class I-related (MR1) proteins, which are specific to antigens such as vitamins. Yet, little is known so far about the activity of CD1d or MR1 proteins. Understanding how to elicit specific effector functions is of great importance to be able to effectively harness these cells therapeutically .
Unfortunately, certain details of cellular organizations and interaction dynamics cannot accurately be determined in the living cell because of the limited spatial resolution of far-field optical fluorescence microscopy, which prevents the discrimination of objects closer together than approximately 200 nm, i.e. details of molecular organization and dynamics on (macro)molecular scales cannot be recovered directly. A remedy to this is recently evolved super-resolution optical microscopy or nanoscopy, and approaches such as STED (stimulated emission depletion microscopy) have now evolved into superior tools for investigating cells and their immunological response at the nanoscale .
We are still missing certain aspects of how CD1d or MR1 are organized within the APC’s membrane are missing. For example, presentation of lipid-CD1d complexes by different types of APCs, binding affinities of CD1d-lipid complexes for the iNKT TCR, loading of CD1d lipid complexes at the cell surface, and incorporation of CD1d-lipid complexes to ganglioside-enriched microdomains in the plasma membranes of APCs have all been shown to influence the functional outcome of iNKT cell activation . In addition, we have just shown strong influence on the functionality and organization of CD1d and MR1 by the cellular cytoskeleton.
The presented project will use novel super-resolution microscopes such as a STED microscope to investigate the cellular (especially the lysosomal and plasma membrane) organization and interaction dynamics of the involved molecules (CD1d, lipids, MR1, vitamins, actin) using advanced optical microscopy tools. We will make use of different cell lines expressing full length or truncated CD1d or MR1 molecules, in the presence or absence of antigen transfer proteins to characterize how loading and distribution of protein-antigen complexes in APCs affects T cell effector function. By using advanced microscopy techniques, we expect to obtain unique new insights into this immunological response process.
This project will be based in the MRC Human Immunology Unit at the Weatherall Institute of Molecular Medicine, with access to state-of-the-art facilities. The project provides an opportunity for training in a broad range of different techniques, such as cell culture, molecular biology, and microscopy, specifically including novel super-resolution microscopy techniques. The disclosure of unique new details of the role of innate sensors such as CD1d or MR1 is an important line of basic immunological research that may translate into new approaches of modulating the immune response during infection and may pave the way to new vaccine adjuvants. Close collaboration with many scientists will be required.
Immunology & T-cell activation, super-resolution STED microscopy, lipid-protein interactions
-  M. Salio, J.D. Silk, E.Y. Jones, V. Cerundolo. Biology of CD1- and MR1-restricted T cells. Annu Rev Immunol, 32, 323-366 (2014).
-  C. Eggeling, K.I. Willig, S.J. Sahl, S.W. Hell. Lens-based fluorescence nanoscopy. Q Rev Biophys 48, 178–243 (2015).
-  J.D. Silk, M. Salio, B.G. Reddy, D. Shepherd, U. Gileadi, J. Brown, S.H. Masri, P. Polzella, G. Ritter, G.S. Besra, E. Y. Jones, R.R. Schmidt, V. Cerundolo. Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells. J Immunol, 180, 6452-6456 (2008).
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