Glycolipid and glycolipid analogue uptake and presentation

The lack of novel adjuvances for human application limits the development of important vaccines. a- galactosylceramide (a-GalCer) and glycolipid analogues represent a group of potential adjuvances. These are taken up by antigen presenting cells (APCs), loaded onto a specific receptor named CD1d and presented to a unique population of natural killer T (NKT) cells, the invariant NKT (iNKT) cells. Upon stimulation, iNKT cells are able to produce large amounts of both pro- and anti-inflammatory cytokines and therefore very interesting targets for adjuvant research and important for the response to various pathogens. The laboratory of Prof. Vincenco Cerundolo studies different promising glycolipid analogues with preferential characteristics for application as vaccine adjuvants. The suggested project aims to clarify the mechanisms of glycolipid and glycolipid agonist uptake using TIRF, confocal laser scanning and electron microscopy. Data will be provided that describe the dynamics of agonist binding to cellular membranes and allow discrimination between receptor mediated uptake and lipid insertion into the membrane for each analogue. Additionally, in human APCs the site of loading onto the receptor CD1d will be determined with a new antibody against a-GalCer loaded CD1d available in the lab of Prof. Cerundolo. Further, the experiments will analyse for the first time the processing of bacterial glycolipids by using Listeria monocytogenes from the laboratory of Prof. Thomas Decker and Escherichia coli which will be loaded with labeled a-GalCer. The uptake and processing as well as the site of CD1d loading will be examined. Besides the important scientific findings the project will help to characterize promising novel adjuvants for human applications and also to evaluate the mouse model for pre-clinical trials.