CD8 T-cell heterogeneity and function: defining the fingerprint of a killer for T-cell therapies Infusion of genetically modified T-cells has produced remarkable clinical outcomes in childhood leukaemia. We aim to examine the process of killing by CD8+ T-cells (CTL) at the molecular level to deliver novel cellular therapies for post-transplant viral infections and haematological cancers. We identified TCR-identical HIV specific CD8+ T-cell clones with distinct suppression of viral replication in vitro, antigen sensitivity, transcriptional signatures and cytokine profiles. In depth functional and molecular characterisation identified that the CTL clone with optimal viral suppression was most antigen responsive with higher expression of previously uncharacterized receptors bearing immunoreceptor tyrosine activating or inhibitory motives (ITAMS/ITIMS) and furthermore did not secrete tumour necrosis factor (TNF). Bisulfite sequencing identified heavy CpG methylation in the TNF core promoter indicating epigenetic modification of CTL effector function post antigen encounter. TCR-identical CTL can therefore be heterogeneous in function and small differences in ontogeny may have significant impacts on cytotoxic efficiency. We aim to identify the transcriptional signature of an “optimal” CTL with high killing efficiency, and identify pathways that can be manipulated to produce populations of CTL enriched with this signature for therapeutic use. This will use a two-pronged approach, using RNA sequencing to examine transcriptomes of ex vivo antigen-specific tetramer sorted CTL populations, combined with in vitro high throughput screening of epigenetic modifiers, drugs targeting known CTL signalling pathways and an shRNA screen focusing on the ITAM/ITIM genes identified above. Our aim is to extend our findings to a high-throughput genome wide ShRNA screen using CD8+ T-cell clones specific for EBV latency II antigens as expressed in 40% of Hodgkins and 20% of DLBC lymphomas. The high-throughput screen will utilise a flow cytometry based cytotoxicity assay at a titrated limiting antigen stimulus, to identify factors that modulate the cytotoxic efficacy of latency II EBV protein- specific CTL clones. Successful hits will be followed up by RNA-sequencing to define the molecular fingerprint of an antigen-experienced CD8+ T-cell with optimal killing efficiency and identify pathways and epigenetic networks that contribute to this state with the aim of producing a therapeutic product for infusion to patients with enhanced chance of cure.