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LEAD SUPERVISOR: Professor Simon J. Davis, Radcliffe Department of Medicine

Co-supervisor: Dr Oliver Bannard, Nuffield Department of Clinical Medicine

 

Commercial partner: MiroBio Ltd / Gilead Sciences, Oxford

Adverse immunological reactions to self and foreign antigens and other situations of abnormal lymphocyte growth are the cause of autoimmune disease, hypersensitivity and asthma, and also lymphoid cancer and transplant rejection. It’s now clear that the decisions that lead to lymphocyte 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, e.g., of 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 In unpublished proof-of-concept experiments, we established a third approach to pathway analysis, wherein the recruitment of fluorescently tagged signaling intermediates to the checkpoint receptors is visualised directly using fluorescence imaging (Fig. 1). 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 model cell surfaces in a semi hi-throughput fashion using confocal microscopy. We propose that a systematic analysis of signaling by three immune checkpoints, 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 signaling 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 signaling 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 signaling pathways already underway. Experiments with pairs of activating and inhibitory antibody agonists will reveal how signals are integrated at microvillar contacts. Bioinformatic analyses at MiroBio Ltd will link the signaling 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 signaling defects and disease susceptibility.

 An image showing recruitment of a signaling intermediate to a ligand-engaged immune checkpoint. In the left panel, microvillar contacts (black dots) are revealed by the loss of fluorescence (gray) in a bilayer presenting the PD-1 ligand PD-L1 and fluorescently tagged large glycocalyx proteins, which are forced out of the contact. The other panelsshow the coincident recruitment of PD-L1 ligands (via PD-1 interactions, cyan) and the downstream signaling protein SHP2 (magenta) to the microvillar contacts.

Figure 1: Recruitment of a signaling intermediate to a ligand-engaged immune checkpoint. In the left panel, microvillar contacts (black dots) are revealed by the loss of fluorescence (gray) in a bilayer presenting the PD-1 ligand PD-L1 and fluorescently tagged large glycocalyx proteins, which are forced out of the contact. The other panels show the coincident recruitment of PD-L1 ligands (via PD-1 interactions, cyan) and the downstream signaling protein SHP2 (magenta) to the microvillar contacts.

In the longer-term, MiroBio Ltd, which is developing antibody agonists to treat autoimmunity but are yet to implement high-level fluorescence imaging, is seeking to use signaling pathway analysis - extended to all immune checkpoints - to help inform target and indication selection. The proposed experiments should create a framework for undertaking systematic analyses of this type. The Oxford laboratory, on the other hand, wants to leverage the bioinformatic strengths of MiroBio Ltd to learn, e.g., how genetic variation influences immune checkpoint signaling outputs.

 

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