The characterization and engineering of human antigen-specific cytotoxic T cells

This D.Phil. thesis focuses on human antigen-specific cytotoxic T lymphocytes (CTLs). In the first part of the thesis, the impact of antigen stimulation strength on CTL activation was investigated by evaluating the global transcriptomes of a wellcharacterized CTL clone and some of its surface protein expressions, with particular focus on immune checkpoint receptors. Based on the gene differentiations, an 'EGR2-CCL1-4-1BB' pathway, which is sensitive to very low-strength antigen stimulation, was identified. We proposed to block CCL1/CCR8 for future cancer therapeutic treatment as this could be a key mechanism for attracting immune-suppressive cells to local tumour-specific CTLs. Further, an âinverse correlation of inhibitory signal and expression levelâ model was developed to explain the expression patterns of five inhibitory immune checkpoint receptors (CTLA-4, PD-1, LAG-3, TIM-3 and TIGIT). These findings throw lights on the understanding of CTL functions and the future design of T cell-based cancer immunotherapy. In the second half the thesis, the potential use of CRISPR/Cas9 technology was explored in manipulating antigen-specific CTLs and the antigen-specific CTL clone gene delivery method was optimised. Successful disruption of PD-1 gene by CRISPR/Cas9 via lentivirus has been achieved on several antigen-specific CTL lines and clones. After disrupting PD-1 gene, CTLs function better when the target cells express high level of PD-1 ligand. This part of work is the first study to knock out PD-1 gene on human antigenspecific primary CD8+ T cells and has the potential for clinical applications such as the treatment of infection, autoimmunity and cancer.