Prof Christian Eggeling and Prof Vincenzo Cerundolo
Many 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 [1-4] (such as proposed here).
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. Understanding how to elicit specific effector functions is of great importance to be able to effectively harness these cells therapeutically.
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 . A specific detail is also the degree of lipid loading of the CD1d in endo-lysosomal compartments of the APC. This loading is enhanced by lysosomal proteins such as saposins and CD1e, which within the lysosome transfer the lipids to the CD1d proteins. Unfortunately, little is known about the details of this lipid transfer and loading process as well as the incorporation into plasma membrane domains, mainly because of the lack of spatial and temporal resolution of live-cell optical microscopy.
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, saposins and CD1e) on < 80 nm spatial scales, so far inaccessible in the living cell by conventional microscopy tools. We will make use of different cell lines expressing full length or truncated CD1d molecules, in the presence or absence of lipid transfer proteins to characterize how loading and distribution of CD1d-lipid complexes in APCs affects iNKT cell effector function. By using superior live-cell microscopy techniques, we expect to obtain unique new insights into this immunological response process.
 C. Eggeling, K.I. Willig, S.J. Sahl, S.W. Hell. Lens-based fluorescence nanoscopy. Q Rev Biophys 48, 178–243 (2015).
 C. Eggeling, K. I. Willig, F. J. Barrantes STED microscopy of living cells - New frontiers in membrane and neurobiology. J Neurochem. 126, 203–212 (2013).
 C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova. V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S.W. Hell Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159-1163 (2009).
 C. Eggeling. Super-resolution optical microscopy of lipid plasma membrane dynamics. Essays Biochemistry 57, 69–80 (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).
 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).
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 by 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 and Molecular, Cell & Systems Biology
Project reference number: 514
|Prof Christian Eggeling||Investigative Medicine Division||Oxford University, Weatherall Institute of Molecular Medicine||GBRfirstname.lastname@example.org|
|Prof Vincenzo Cerundolo||Investigative Medicine Division||Oxford University, Weatherall Institute of Molecular Medicine||GBRemail@example.com|
There are no publications listed for this DPhil project.