Cytotoxic CD8+ T Cells (CTL) play a key role in the host response to hepatitis C virus (HCV) infections, contributing to viral clearance and protective immunity. The characterisation of the HCV-specific CTLs during viral infection can therefore reveal key mechanisms that underpin the determinants of effective immunity. Upon antigen stimulation, activated CTLs proliferate and differentiate, ultimately generating heterogeneous progeny. The advent of single-cell sorting and RNA-seq has enabled the analysis of these heterogenous populations directly from human samples. In this study, we examined the transcriptome profile of HCV-specific CTLs isolated from a subject with chronic HCV infection at the single-cell level. CTLs specific for a HLA-I A0201 restricted HCV epitope, CINGVCWTV were obtained from peripheral blood mononuclear cells (PBMCs), and used to generate a cell line. We applied single-cell RNAseq to cells derived directly from patient’s PBMCs and compared to those grown ex vivo, with and without stimulation by autologous peptides. We analysed the difference between the three groups of single cells using a list of genes previously associated with CTL functions. Genes associated with cytotoxic response (IFN-g, perforin, granzyme B) were highly expressed in cells grown ex vivo, but not in PBMC-derived CTLs. Using unsupervised clustering analysis, we identified co-expression clusters within each group. By filtering out the cell-cycle related genes (a major co-founder), we revealed that restimulated cells showed a high level of heterogeneity when compared to unstimulated (p-value <0.0001). The chemokine cell receptors (CCL3, CCL4, CXCR6) were among the most variable genes, while enrichment analysis identified IRF1 and IRF7 as key transcription factors differentiating stimulated and unstimulated cells. Ongoing analyses will investigate the relationship between transcriptomic and phenotypic data from CTL surface markers of differentiation, activation and exhaustion using flow cytometry. This will provide us unprecedented opportunity to exploit genotype-phenotype relationship in antigen-specific T-cells