Dynamic tracing of T cell receptor signalling

Elliot, Thomas Ashton Emmerson ORCID: 0000-0001-9292-2472 (2024). Dynamic tracing of T cell receptor signalling. University of Birmingham. Ph.D.

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Abstract

The T cell receptor (TCR) determines the antigen-specificity of T cells and tightly controls T cell activation status. The TCR can be ligated by binding to peptide loaded MHC, initiating a complex set of downstream signalling pathways, that result in nuclear entry of transcription factors that regulate the expression of genes involved in T cell function. TCR signalling can lead to proliferation, migration, cytokine production, metabolic re-programming, and killing of target cells. The TCR is co-regulated by a number of co-stimulatory and co-inhibitory receptors; expression of TCR co-receptors changes dramatically upon T cell activation. TCR signalling is therefore a very dynamic process regulated by a number of context dependent factors. To study dynamic control of TCR signalling we generated Nur77 Tempo mice, that express a Fluorescent Timer protein under the control of the regulatory regions of Nr4a1, the expression of which is dependent on TCR signalling. Fluorescent Timer protein matures over time, shifting its emission spectrum from blue to red. This allows for distal reporting of TCR signalling events with high time sensitivity. We validated the utility of Nur77 Tempo to track TCR signalling events and compared it to a similar reporter, Nr4a3 Tocky, that reports Fluorescent Timer under the regulation of related gene Nr4a3. We highlight key differences in the expression patterns of Nur77 Tempo and Nr4a3 Tocky, notably that Nr4a3 Tocky requires a higher strength of TCR signal and is absolutely dependent on Calcineurin-NFAT signalling. In contrast, Nur77 Tempo can detect weaker signals such as those involved in thymic positive selection and is dependent on NFAT independent signalling pathways. Using a tumour model and an in vitro model of T cell exhaustion, we show that chronic TCR stimulation of CD8\(^+\) T cells results in sustained NFAT signalling but shutting down of NFAT independent signalling in a way that is not reversible with immunotherapy. We go on to show that transient inhibition of NFAT signalling is sufficient to partially reverse some phenotypic features of CD8\(^+\) T cell exhaustion.
In a separate body of work, we investigate the drivers of intra-tumoural immunity in human colorectal cancer (CRC) patients. A small subset of CRC patients have an aggressive CD8\(^+\) T cell mediated immune response due to a high neo-antigen burden as a result of microsatellite instability and abundant frameshift and missense mutations. However, some patients without microsatellite instability have transcriptional scores of anti-tumour immunity that are equivalent to those that do. We use CD39, a biomarker of tumour antigen reactive T cells, to evaluate the role of antigen recognition in driving high immune scores, finding a redundant role among microsatellite stable patients. We went on to investigate the potential role of microbial adjuvantisation. Meta-genome sequencing was not able to identify differences in relative bacterial abundance between low and high immunity microsatellite stable patients. However, many genes involved in innate immune response to bacteria were elevated in high immunity patients, particularly some transcripts encoding guanylate binding proteins. We confirmed protein expression of GBP5, a necessary component of NLRP3 inflammasome assembly. We confirmed that human intestinal epithelial cells can clave NLP3 executor Caspase-1 and posit a GBP5-NLRP3-IL-18 pathway as a key regulator of immunity in low mutational burden CRC

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):