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Unravelling cross-presentation pathways using a chemical biology approach

Periodic Reporting for period 3 - Crosstag (Unravelling cross-presentation pathways using a chemical biology approach)

Reporting period: 2018-05-01 to 2019-10-31

Immune therapies are therefore currently being pursued to reinvigorate the immune reaction against tumours. This is not trivial, as the right cytotoxic T-cells must be activated against a tumour-specific antigen to get the right response; all in the face of a tumour attempting to switch the resulting immune response back off.

One method for enhancing the cytotoxic anti-tumour response is by targeting receptors on certain immune cells that can result in the enhanced activation of these cytotoxic T-cells. One of these is the mannose receptor. However, this glycoprotein-binding receptor appears to have two functions: it can either potently enhance cytotoxic T-cell activation, or, in certain cases, reduce the activation of these T-cells when a vaccine is targeted towards it. Current tools, such as anti-MR antibodies and randomly glycosylated ligands fail to selectively enhance cytotoxic T-cell activation, as it appears that the receptor can distinguish between subtle differences in sugar coatings on the proteins it enhances.

The main aim of this proposal is to determine what structural parameters of the glycoprotein antigen result in this enhanced cytotoxic T-cell activation. Furthermore, using new imaging techniques based on super resolution microscopy, it is the aim of the proposal to look at how the cells activating the cytotoxic T-cells treat vaccines differently in case of these different vaccines.

The outcome of this proposal will hopefully be an improved understanding of how we can enhance this cytotoxic T-cell response. This, in the face of the recent breakthroughs in cancer immunotherapy, will lead to better, cheaper immunotherapies for cancer.
The aim of the project is therefor two-fold:
On the one hand a library of biologically traceable single glycoform ligands are being synthesised that target different aspects of the biology of the mannose receptor. - with controlled variation in glycan nature, stoichiometry and positioning - for the MR and study differences in uptake, routing and antigen presentation.

So far we have made a series of different antigens that contain bioorthogonal groups for imaging. We have made some cancer long peptide vaccines, as well as model protein antigens, and proteins related to auto-immune emergence. We have also made a series of carbohydrates targeting different binding domains of the carbohydrate receptors and constructs of these antigens modified with some of these sugars. We are now evaluating the immunological properties of these constructs.

The 2nd aim is to develop a new imaging technique that will allow the subcellular tracking of the antigens as it being handled differently by the receptors. For this we have now developed an approach that allows the super-positioning of a super-resolution microscopy image of the bioorthogonal antigens on an 75 nanometer thin section of a cell and place it on top of an electron microscopy image of this same thin section. This way we can get the most information on the location of antigen as it is being processed in the cell.
We have progressed beyond the state of the art and the proposal w.r.t. the imaging method. The initial plan was to only look at super-resolution images of the antigen. However, by now being able to combine it with electron microscopy, we not only obtain information on the antigen, even as it is being degraded, we can place it in the context of the organelles in the cells where these antigens are found.

By the end of the project, I hope to have met all goals, and imaged the different routes that antigens take depending on the glycan-mannose receptor interaction; both for cancer vaccine antigens, as well as for auto-immune antigens.
A correlative image of a dendritic cell showing vaccine processing enzymes in action