Imaging-based analysis of signalling pathways triggered by immune checkpoint receptors
LEAD SUPERVISOR: Prof. Simon Davis, Radcliffe Department of Medicine
Co-supervisor: Dr Oliver Bannard, Nuffield Department of Medicine
Commercial partner: Gilead Sciences (Oxford)
It’s now clear that the decisions that lead to lymphocyte activation, survival or death are decided not just by antigen binding but also by activating or inhibitory (“checkpoint”) receptors that provide positive or negative selection signals and tune cells to vital cues in their environment. Understanding these processes has led to the development of blocking antibodies that prevent signalling by masking the ligands of inhibitory receptors, an approach that has transformed cancer immunotherapy. The reverse approach, of trying to agonise the immune checkpoints with antibodies in the context of, e.g., autoimmunity, has only recently been tried for two targets. New work from the Davis laboratory has revealed how blocking and agonistic antibodies differ and shown that all the immune checkpoints could, in principle, be agonised. The problem is that there are ~70 of these receptors and it’s unclear which of the checkpoints should be tried most urgently. Informed choices cannot be made because so little is understood about the signalling pathways used by checkpoint receptors, and how the checkpoints are differentiated one from another.
One approach to pathway analysis is based on immune-precipitation or “pull-downs”, but important low affinity interactions may be lost during wash steps. Alternatively, individual domain interactions can be analyzed directly, but likely cooperative effects will go undetected. In unpublished proof-of-concept experiments, we have established a third approach to pathway analysis, wherein the recruitment of fluorescently-tagged signalling intermediates to the checkpoint receptors is visualised directly using fluorescence imaging. This was made possible by our discovery that T-cell fate decisions are made at large numbers of small ‘microvillar’ contacts formed by T cells with apposing surfaces, that can be visualized on “second-generation” supported lipid bilayer-based model cell surfaces (Jenkins et al. 2023 Nature Comms 14, 1611) in a semi hi-throughput fashion using confocal microscopy. We propose that a systematic analysis of signalling by three immune checkpoints, i.e., PD-1, BTLA and TIGIT, and a fourth, activating receptor, the T-cell receptor, should be undertaken. We will determine, in Oxford, which of 66 possible signalling proteins expressed by T cells are recruited to these receptors at microvillar contacts following triggering with agonistic antibodies. Comparisons of the ‘hits’ will provide the first insights into the extent to which inhibitory signalling pathways differ from one another and overlap with pathways triggered by activating receptors. Arrayed CRISPR screens will be used to test the hits and identify co-operative interactions underpinning receptor recruitment, complementing whole-genome screens of the signalling pathways already underway. Experiments with pairs of activating and inhibitory antibody agonists will reveal how signals are integrated at microvillar contacts. Bioinformatic analyses at Gilead Sciences (Oxford) will link the signalling pathways to disease indications, using publicly available and other genetic (e.g., GWAS) data. The testing of genetic variants in the imaging experiments will establish their contribution to signalling defects and disease susceptibility.
In the longer-term, Gilead, which is developing antibody agonists to treat autoimmunity but are yet to implement high-level fluorescence imaging, is seeking to use signalling pathway analysis – extended to all immune checkpoints – to help inform target and indication selection. The proposed experiments should create an experimental framework for undertaking systematic analyses of this type. The Oxford laboratory, on the other hand, wants to leverage the bioinformatic strengths of Gilead to learn, e.g. how genetic variation influences immune checkpoint signalling outputs.
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