Cell Type-Specific Analysis of Immune Checkpoint Signalling Networks Underpinning Cancer Immunotherapy

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Immune checkpoints are proteins expressed on the surfaces of immune cells that suppress their activity (normally so that they don't attack us). Work on just two immune checkpoints has revolutionised cancer therapy by producing durable responses in previously untreatable diseases such as melanoma, by blocking the suppressive effects of the receptors so that the leukocytes are free to attack tumours. But there are as many as 60 immune checkpoints regulating the immune system, underscoring the extraordinary scope for medical intervention via the checkpoints, and emphasising how much work is still to be done. Remarkably, despite their enormous significance, very little is known about how the immune checkpoints work, i.e. the molecular pathways they use to switch off immune responses to tumours. In this proposal we aim to explore how immune checkpoints differ with regard to the molecular mechanisms of their activity in three major types of immune cells (i.e. B cells and T cells, and myeloid cells), and to learn whether it will be possible to exploit these differences therapeutically. If it turns out that the immune checkpoints invoke the same pathways, it is unlikely that we will be able to make them work better collectively. However, based on what is already known about these pathways, this seems very unlikely to be the case. We will start our study at the level of a model two-cell co-culture system in vitro (i.e. in "test tubes"), where we will be able to test multiple approaches. To study what happens in the setting of authentic tumours, we will create three-dimensional (3D) cultures of actual tumours, which we can study in the course of their responses to immunotherapy. But the main problem with understanding how the immune checkpoints work is that our current knowledge of human cellular signalling pathways is very incomplete and highly biased to well-studied ones. For example, 30-50% of the targets for the most important groups of enzymes driving signalling, called kinases and phosphatases, are completely unknown. This suggests that important pathways and processes may currently be undiscovered. Limiting our studies of immune checkpoint signalling to the known pathways would reveal only part of the jigsaw and would mean that effective new ways to treat cancer might be wholly overlooked. To circumvent this issue, we are proposing to use a strategy that combines genetic perturbations, i.e. "gene knockouts" of all the possible kinases and phosphatases that could be involved in the signaling pathways in the immune cells under study, with measurements of signalling outcomes based on a convenient, manageable set of signaling pathway elements we can easily and accurately measure in single cells (a great leap forward). Our goal is to be able to use this small set of pathway elements to build out to the complete network. To do this we will be developing new computational pipelines in order to obtain comprehensive and accurate pictures of the whole signalling network, for each of the main sets of leykocytes involved in anti-tumour responses. Once we show that the new pipeline works, we will be able to compare and contrast how immune checkpoints vary and how different types of blockade of these proteins alters the activities of the immune cells attacking cancers. We're very confident that our work will plug major gaps in our basic understanding of immune checkpoints which will be of considerable interest to all immunologists. But more importantly, our work could suggest important new ways to improve immune checkpoint blockade cancer immunotherapy.