Periodic Reporting for period 3 - KineTic (New Reagents for Quantifying the Routing and Kinetics of T-cell Activation)
Reporting period: 2023-08-01 to 2025-01-31
The behaviour of T-cells underpins all essential processes in the immune system, ranging from fighting pathogens to the clearance of tumours. Their behaviour can, however, also be highly pathogenic: aberrant T-cell behaviour is involved in cancer emergence and persistence, and in the pathogenesis of many auto-immune diseases. Understanding the behaviour of these cells is therefore essential for understanding the emergence of these diseases, as well as to understand the curative potential of T-cells.
Despite these critical roles in immunology, many of the molecular fundamentals of T-cell activation and the effect this has on their behaviour in pathogen and tumour clearance remain unknown and my career goal is to provide new chemical methodology to better understand the activation mechanisms of T-cells at the molecular level. With this, I hope to support improvements in the spate of revolutionary, yet highly expensive, personalized T-cell-based therapies that are in clinical and pre-clinical development. To date their success has been rather capricious in the making: T-cell activating agents have proven curative in a small number of tumours, but their broad application to all tumours types has not yet materialized, with their failures in the treatment of solid tumours particularly notable. Understanding the basic physiology of T-cell activation, deactivation and therapeutic reactivation is therefore of huge fundamental and clinical importance.
This ERC Consolidator Grant was written to look at two aspects of fundamental T-cell activation biology, namely the speed at which certain processes occur during the activation of the cytotoxic T-cells, and what organelles are critically involved in the routing and processing of antigen during cytotoxic T-cell activation. The inspiration for this proposal was twofold. First, T-cells are activated by dendritic cells (DCs) in the lymph node. These cells, however normally reside in the tissues all over your body and only travel to the lymph node when they have encountered something dangerous to present to the T-cell. During their trip to the lymph node they will degrade the material they encountered and present short peptide from the disease-causing agent on their surface. They also present an activating signal. If a cytotoxic T-cell can bind to the bits of the pathogen, and receives the danger signal, it becomes a highly potent killer cell, capable of killing every cell infected with the pathogen.
The conundrum in this process lies in the timing: the processing and presentation is very fast (and already begins at 15 minutes or so after uptake). The lifetime on the surface of the pathogen is also very short (usually a few hours). Yet, what is so surprising is that the travel of the DC to the lymph node takes as much as three days! This means that, if current theories are correct and complete, all foreign peptides should have vanished from the DC by the time it reaches the lymph node. As this is clearly not the case, there must be other mechanisms at play here. The KineTic proposal was written to develop new chemistry-based method to study the timing of these processing processes in such a way that we can figure out how long things take in a mouse in a real immune activation event. With this we hopefully shed light on the potential solution to this conundrum.
The second inspiration for the chemistry that we are developing revolves the route the antigen takes. Much controversy remains regarding how the material from the pathogen moves through the cell on the way to the surface where it can activate the cytotoxic T-cells. This has been particularly difficult to study as the material is cut into short peptide pieces during the process. This makes them very hard to find.
The aim of the proposal was therefore twofold: to develop new chemistry to measure the speed of antigen processing in vivo. And to develop new chemistry that would allow us to determine what the role of specific organelles was in the route map that the antigen takes within the dendritic cell. Again, preferably in such a manner that we could eventually do it in mice.
Despite these critical roles in immunology, many of the molecular fundamentals of T-cell activation and the effect this has on their behaviour in pathogen and tumour clearance remain unknown and my career goal is to provide new chemical methodology to better understand the activation mechanisms of T-cells at the molecular level. With this, I hope to support improvements in the spate of revolutionary, yet highly expensive, personalized T-cell-based therapies that are in clinical and pre-clinical development. To date their success has been rather capricious in the making: T-cell activating agents have proven curative in a small number of tumours, but their broad application to all tumours types has not yet materialized, with their failures in the treatment of solid tumours particularly notable. Understanding the basic physiology of T-cell activation, deactivation and therapeutic reactivation is therefore of huge fundamental and clinical importance.
This ERC Consolidator Grant was written to look at two aspects of fundamental T-cell activation biology, namely the speed at which certain processes occur during the activation of the cytotoxic T-cells, and what organelles are critically involved in the routing and processing of antigen during cytotoxic T-cell activation. The inspiration for this proposal was twofold. First, T-cells are activated by dendritic cells (DCs) in the lymph node. These cells, however normally reside in the tissues all over your body and only travel to the lymph node when they have encountered something dangerous to present to the T-cell. During their trip to the lymph node they will degrade the material they encountered and present short peptide from the disease-causing agent on their surface. They also present an activating signal. If a cytotoxic T-cell can bind to the bits of the pathogen, and receives the danger signal, it becomes a highly potent killer cell, capable of killing every cell infected with the pathogen.
The conundrum in this process lies in the timing: the processing and presentation is very fast (and already begins at 15 minutes or so after uptake). The lifetime on the surface of the pathogen is also very short (usually a few hours). Yet, what is so surprising is that the travel of the DC to the lymph node takes as much as three days! This means that, if current theories are correct and complete, all foreign peptides should have vanished from the DC by the time it reaches the lymph node. As this is clearly not the case, there must be other mechanisms at play here. The KineTic proposal was written to develop new chemistry-based method to study the timing of these processing processes in such a way that we can figure out how long things take in a mouse in a real immune activation event. With this we hopefully shed light on the potential solution to this conundrum.
The second inspiration for the chemistry that we are developing revolves the route the antigen takes. Much controversy remains regarding how the material from the pathogen moves through the cell on the way to the surface where it can activate the cytotoxic T-cells. This has been particularly difficult to study as the material is cut into short peptide pieces during the process. This makes them very hard to find.
The aim of the proposal was therefore twofold: to develop new chemistry to measure the speed of antigen processing in vivo. And to develop new chemistry that would allow us to determine what the role of specific organelles was in the route map that the antigen takes within the dendritic cell. Again, preferably in such a manner that we could eventually do it in mice.
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(opens in new window)). 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(opens in new window)). 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(opens in new window)). 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(opens in new window)). 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(opens in new window)). 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(opens in new window)) and also that of fatty acids (https://onlinelibrary.wiley.com/doi/full/10.1002/ange.202207640(opens in new window)).
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.
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(opens in new window)). 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(opens in new window)). 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(opens in new window)). 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(opens in new window)). 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(opens in new window)). 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(opens in new window)) and also that of fatty acids (https://onlinelibrary.wiley.com/doi/full/10.1002/ange.202207640(opens in new window)).
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.
The KineTic project has so far yielded many tools that offer a significant advance to the field of immunology. The tools that for the first time allow the study of antigen processing and presentation from the moment of uptake to the activation of T-cells without affecting structure and antigen behaviour are completely new. Before the end of the grant, we will have developed a detailed route-map of the antigen in this manner, correlating in which organelle an antigen accumulated to the success of a subsequent immune response.
With regards to the study of the timing of these processes, we will have the complete toolkit and first in vivo timing studies attempted before the end of the grant. The in vitro system is ready for writing up, and the first system for using the chemistry to activate only antigens passing through the lysosome is currently under review. These systems were the main deliverables of the proposal.
In addition, we will have also produced the aforementioned imaging approach and the toolkit to study nutrient fluxes during T-cell activation.
With regards to the study of the timing of these processes, we will have the complete toolkit and first in vivo timing studies attempted before the end of the grant. The in vitro system is ready for writing up, and the first system for using the chemistry to activate only antigens passing through the lysosome is currently under review. These systems were the main deliverables of the proposal.
In addition, we will have also produced the aforementioned imaging approach and the toolkit to study nutrient fluxes during T-cell activation.