Analysis of lipid-antigen assisted T-cell activation using live-cell super-resolution (STED) microscopy
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. Project 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.
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.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The Department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.
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