Investigating T cell signalling dynamics using light-controllable chimeric antigen receptor

T cells are a part of the immune system that recognise infected cells and elicit an immune response to remove them from the body. T cells traverse between the lymph nodes and blood looking for antigen-presenting cells (APCs). APCs present peptides derived from foreign pathogens or infected cells to the TCR via MHC molecules. T cells scan the surfaces of all APCs present in the lymph nodes at a high velocity and yet specifically bind to the cognate peptide-MHC with a remarkable sensitivity to discriminate between healthy and infected cells. T cells need to generate fast response against targets but also need the response to be specific. It is hypothesised that this trade-off between the requirement of specificity and fast response is managed by integrating signals from multiple interactions with APCs to generate a robust immune response. T cells can only integrate the signals from multiple interactions if they retain an imprint of each interaction in the activation network following the engagement of the TCR to the cognate pMHC. To quantify the capacity of T cells to integrate stimuli from multiple interactions, I have used a light-controllable synthetic receptor, called Optogenetic chimeric antigen receptor (OptoCAR). OptoCAR allows precise, tunable, and reversible activation of T cells controlled by the blue light of wavelength 450 nm and binds to a specific antigen on APCs without a need for antigen presentation by MHC molecules. I implemented the OptoCAR system in Jurkat cells, a T-cell cancer cell line to first quantify the persistence of receptor signalling information in downstream signalling nodes of the T cell activation pathway and found the information at the transcriptional factor level is dissipated within 15 minutes of TCR signal cessation at the cell surface and the cells require persistent signalling to drive gene transcription. Utilising pulsatile trains of signalling, I quantified the T cells can only accumulate the memory of individual interactions with APCs if the intervals between the interactions is within a window of ∼10 minutes. Following on from this result, I implemented the Opto19CAR system that recapitulates the structure and function of CD19CAR used in cancer immunotherapy but can be optically controlled in human primary CD4+ T cells to investigate the signalling dynamics that drive the development of dysfunctional exhausted T cell fate when stimulated persistently by antigens such as in case of cancer. T cell exhaustion characterised by the increase in the expression of immune response suppressing inhibitory receptors is one of the major causes of failure in non-responding or relapsed patients treated with CD19CAR T therapy where the patient’s T cells are engineered with CARs to bind and respond to CD19 antigens expressing cancerous B cells. By using the light-controllable Opto19CAR, I modulated the receptor stimulation parameters to provide the T cells with pulses of signals instead of the continuous signal and found that this decoupled the expression of activation-associated response and exhaustion-associated response in CD4+ T cells: activation response favoured pulsatile stimulation of the receptor whereas strong exhaustion response was generated by continuous stimulation of the receptor. This decoupling of the activation and exhaustion-associated response was observed at both the protein and gene expression levels of the T cell activation networks. This mechanistic understanding of the development of T cell exhaustion will help to mitigate it and improve current CAR T therapies