Understanding the regulation of cytotoxic T lymphocyte signalling and activity through single-cell genomics

Cytotoxic T lymphocytes (CTLs) are a specialized type of white blood cell that play an important role in combating viral infections and cancer. In fact, several new cancer treatments focus on improving the response of these cells to fight tumours. Like many cells in the body, CTLs are very sensitive to their surroundings, altering their responses based on the other cells and signalling molecules around them. Thus, development of drugs that alter the function of these cells can be difficult. The first step in such a drug development process is understanding how these cells behave in different contexts. Beneficially for immune responses, but confusingly for researchers, the internal signalling events of CTL responses are extremely rapid, on the order of minutes. Thus, within a group of CTLs, each cell may be at a slightly different stage in its response and exhibit different characteristics. Until recently, technologies used to examine changes in these cells required that thousands be pooled together. The measurement was therefore an average across the whole pool of cells and didn't reflect the precise response stage of each individual cell. New technologies now enable measurements to be made in single cells, allowing a huge amount of information to be gathered about how individual cells behave and about the fine-tuned steps of their responses. In this project, we propose to examine single CTLs in various contexts relevant to their responses to cancer, including initial activation and in the presence of immune-enhancing drugs. We are particularly interested in highlighting potential drug targets and in improving methods to analyse this new type of data.

Cytotoxic T lymphocytes (CTLs), which develop from naïve CD8+ T cells, are one of the central players in immune defence against cancer. In recent years, these cells have become a key target for therapies that enhance the anti-tumour immune response. T cell activation is a highly regulated but rapid process, requiring a multitude of coordinated signalling events to ensure fast, specific responses to antigen. While the transcriptomics of these early signalling processes have been studied in bulk cellular populations, many events in early activation are obscured by averaging across a heterogeneous cell pool. We here propose to use single-cell sequencing technologies to examine the transcriptome and coordinated protein expression during activation of CD8+ T cells. Specifically, we aim 1) to construct a fine-grained time-course of signalling during early T cell activation, 2) to understand how TCR affinity affects pathway usage and intercellular heterogeneity in early responses, 3) to understand how check-point molecules and their inhibitors affect the CTL transcriptome, 4) to create a pipeline for integrated analysis of transcriptomic and surface protein expression data from single cells. We will use a murine model of transgenic CD8+ T cells specific for a chicken ovalbumin peptide to have fine-tuned control over T cell activation. Single cells will be index sorted into lysis buffer by fluorescence-activated cell sorting to record surface protein information. Transcriptomic profiles will be generated by RNA sequencing. Starting from existing analysis methods for differentially distributed genes, we will develop tools to integrate flow cytometry and transcriptomic data to better characterise activation states and signalling pathways. This work will shed light on signalling events in T cell activation and response to environmental stimuli. Such information is crucial to finding targets for novel therapeutics.

The proposed research has the potential to benefit several sectors outside of the academic research community. First and most simply, I plan to communicate the research topic to the interested public through the Cambridge Science Festival to help broaden public knowledge of the work that academics are carrying out in the fields of immunology and statistics to benefit cancer research. It also allows the public the opportunity to ask questions about science they have heard in the news or in conversation, which can increase both understanding of and confidence in the work scientists are doing. Second, by furthering my career goals to become an academic group leader, this Skills Development fellowship could contribute to the knowledge and skills of future students I would teach and supervise. In addition, in the long term, this research has the potential to identify a target for development of a novel drug or suggest stratified use of a current therapeutic. This would first have the potential for economic benefit to the Cancer Research UK Cambridge Institute and greater University of Cambridge through patenting and commercialisation. Second, this would impact NHS Cambridge University Hospitals if a proof-of-concept study or clinical trials were initiated. Most importantly, this research has the potential to improve health by contributing to development of an additional treatment option for cancer patients.