The first part of this chemical diptych was the development of live cell compatible chemistry that would allow us to control and study the timing of antigen processing and presentation. For this, we used a chemical method that we had previously developed, that allowed us to chemically control the recognition event between a dendritic cell presenting a peptide and a T-cell that can recognize that particular peptide with chemical switches. We first spent considerable time trying to modify these reagents, so the chemistry could be applied to the study of vaccine like constructs, rather than very short model peptides that behave differently in the biological system. This required us to come up with new, rather leftfield, synthesis strategies. The manuscript for this work should see the light of day in 2023.
This live cell (and whole mouse compatible) chemistry, combined with these new vaccine-reagents, now allow us to give a vaccine at a point in time, and ‘activate’ the peptides that are on the cell surface at any given point in time after the vaccination. This method is allowing us to now study (in a dish first, and soon in a mouse) how fast and how persistent this process is. But also whether there are any differences in vivo in how long a product remains on the cell surface. This will hopefully give us refreshing new insights in the antigen timing conundrum. We have one manuscript covering this work under review at present, and have an additional one being readied for submission.
For the second part of the project – in which we aim to follow the routing of an antigen inside a dendritic cell – we have also developed new methodology first that we are currently applying to the study of the subcellular route of the cytotoxic T-cell-activating peptides. The first method to do so used click chemistry: antigens (peptide, protein or whole cell) were modified with click-reactive groups and followed during their path inside an immune cell (first paper:
https://pubs.rsc.org/en/content/articlehtml/2021/cb/d1cb00009h(odnośnik otworzy się w nowym oknie)). We used, for example, a new method called bioorthogonal-correlative light-electron microscopy in which the click signal from the antigen is positioned on an electron micrograph of the same cell and can tell us in which organelle it resides (
https://pubs.acs.org/doi/full/10.1021/acscentsci.0c00539(odnośnik otworzy się w nowym oknie)). We were the first in the world to develop this method, and the first to combine it with super-resolution microscopy. In addition, we have come up with another new method to study antigen degradation with click chemistry. Using a new background-free retrieval method to pull the partially degraded antigen from a dendritic cell lysate we can quantify how it has been degraded (
https://doi.org/10.1002/cbic.202300082(odnośnik otworzy się w nowym oknie)). This allows us to study the speed and nature at which this degradation occurs under different conditions in vivo.
Aside from these two main research lines, we have had some collateral successes resulting from this Consolidator Grant. In collaboration with Ton Schumacher at the NKI, we have made a new chemical reagent that allows us to ‘tag’ individual T-cells in primary human tumour material for downstream sequencing (
https://www.nature.com/articles/s41589-021-00839-x(odnośnik otworzy się w nowym oknie)). Together with Lorenzo Albertazzi of the TU-Eindhoven, we have also started looking at the biochemistry of uptake of antigen and the speed at which this occurs. For this we developed a completely new method that, for the first time, allows us to quantify the on and off rates of weak binding events on the surface of a living cell (
https://www.nature.com/articles/s41589-021-00896-2(odnośnik otworzy się w nowym oknie)). A third collateral breakthrough, this time from a collaboration with Linda Sinclair (University of Dundee) and David Finlay (Trinity College Dublin), occurred from a control compound. We found that we could use clickable metabolites to study changes in the nutrient fluxes by immune cells that are being activated. In this manner we could image glutamine uptake activity in primary immune cells (only available as preprint at present:
https://www.biorxiv.org/content/10.1101/2022.09.29.510040v1.abstract(odnośnik otworzy się w nowym oknie)) and also that of fatty acids (
https://onlinelibrary.wiley.com/doi/full/10.1002/ange.202207640(odnośnik otworzy się w nowym oknie)).
All these mainline and collateral successes are helping me take small steps towards my career goal of understanding the T-cell activation biology at the single molecule level.
