Virus-specific CD8+ T (CTL) cells play a pivotal role in protection from viral infection. Several viruses infecting humans, such as HIV, and hepatitis C virus (HCV) develop chronic infection, which are characterized by both immune escape and CTL cell exhaustion. Despite recent developments, how immune escape and exhaustion develop, the precise role of TCR diversity in controlling immune escape, and the overall differentiation pathways of CTL responses remains unclear. Better understanding of how T cell responses develop is timely needed for development of T cell based vaccines that can provide long term immune protection. Single cell technologies are now significantly improving the understanding of these highly dynamic and heterogeneous populations of cells.

In this presentation I will review our recent studies on unique longitudinal samples of primary HCV infection using multicolor flow cytometry for phenotypic characterisation of Ag-CTLs, along with single cell transcriptomic and TCR diversity analyses. These studies revealed several differentiation pathways occurring in subpopulation of CTL within the same subject, with majority of responses dominated by terminally differentiated effector memory cells (CCR7^low CD45RO^high, KLRG1^highCD127^low) during the first 6 months of infection, with an early and elevated expression of multiple co-inhibitory markers (PD-1, KLRG1 and 2B4) targeting both conserved as well as escape HCV variants. There was an association of viral escape with the magnitude of the CTL response and ongoing diversification of TCR clonotypes, suggesting ongoing evolution of CTLs in response to prolonged viral exposure. Future directions involve optimization and cost reduction of single cell transcriptomics and their application to a broader range of diseases.