Imaging-based analysis of signaling pathways triggered by immune checkpoint receptors

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 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.

We have recently shown that the recruitment of fluorescently-tagged signaling intermediates to checkpoint receptors can be visualised directly using fluorescence imaging in a semi hi-throughput fashion using confocal microscopy.

We propose that a systematic analysis of signaling by three immune checkpoints, i.e., PD-1, BTLA and TIGIT, and a fourth, activating receptor, the T-cell receptor, should be undertaken.

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.