Novel fluorescent probes for monitoring T cell localisation and activity
Item statusRestricted Access
Embargo end date19/05/2021
Scott, Jamie Iain
Cancer is one of the deadliest diseases known to humanity. In the UK someone is diagnosed with cancer every two minutes. The associated mortality rates vary depending on the type of cancer but despite this being such a prevalent problem in modern history we still have a limited understanding of mechanisms behind cancer and how best to treat it. One of the main complications with cancer is the malfunctioning response of the human immune system. This can occur due to the adaptive immune system, particularly T cells, being unable to detect cancer cells in the body thus avoiding a competent immune response; or the tumour site itself being able to manipulate immune cells into a state of exhaustion and inactivity. Over the last decade therapies aimed at enhancing T cell responses have come to the fore-front of cancer treatments. They include post adoptive transfer, a technique which confers T cells to recognise cancerous cells, and immune checkpoint inhibitors which aim to reinvigorate the T cell response following initial immune suppression. However, there are certain drawbacks associated with each type of therapy. In the case of post adoptive transfer, regulatory approval is deemed difficult due to the off-target toxicity of the enhanced cells. With immune checkpoint inhibitors there is a problem with suitable choice of therapy as each cancer type and patient has different responses. Currently there are no suitable biomarkers of reinvigoration, which slows therapy choice and evaluation. Consequently, there is also a need to establish an assay to screen for new biologics or small molecules that can boost the immune response. In this thesis, fluorescence microscopy was utilised to address both issues through long term in vivo tracking of CD4+ T cells following post adoptive transfer and through the use of peptide-based fluorogenic probes to assess the activity of cytotoxic CD8+ T cells via granzyme B activity. Herein was developed two novel chemical technologies: 1) a NIR tricarbocyanine dye, CIR38M, that is able to track over time smaller numbers of T cells than current NIR agents, and to visualize antigen-driven accumulation of immune cells at specific sites in vivo and 2) probe H1, a peptide based fluorogenic probe that can image and distinguish between activated and non-activated CD8+ T cells through granzyme B activity. H1 was also utilized to create a high throughput assay to establish the efficacy of potential immune stimulating agents and demonstrated its ability to identify CD8+ T cell mediated tumour regression via granzyme B activity in models of mouse squamous cell carcinoma. These chemical technologies will improve longitudinal imaging studies and help to establish new immune stimulating therapies as well as to assess the efficacy of current immunotherapies in murine models with potential for human translation.