Linking functional phenotype, gene expression profile and clonotype diversity of T cells during a viral infection
S. Rizzettoa,b, A. Elthalaa,b, M.Rasolia,b, E. Keoshkeriana,b, K. Wingc, D. Buschc, A.R. Lloyda,b, R.A. Bulla,b, F. Luciania,b
aSchool of Medical Science, UNSW, Australia; bKirby Institute, Australia; cInstitute for Medical Microbiology, TUM, Munich, Germany
CD8+ T (CTL) cells play a key role in protection from viral infection. Better understanding of the heterogeneous phenotypes of T cell subsets as they evolve during an immune response is timely, given their relevance to the development of T cell vaccines that may provide robust long-term immunity. We have developed a single cell approach to identify and link functional phenotype, gene expression profile, TCR diversity and affinity for specific epitope. This analysis was performed in blood samples from HCV infected subjects followed longitudinally from pre-infection until disease outcome (clearance or chronicity).
Antigen-specific CTLs were identified via autologous epitope discovery and functional (IFNγ-ELISPOT) validation. Index sorting was utilised to link surface immune-phenotyping with gene expression profile, and the full length TCRαβ repertoire was identified via single cell RNAseq using novel tools (1).
Here we report unpublished data from one subject that cleared HCV.A unique autologous CTL responses was found carrying a single TCR, associated to high IFNγ production, and with high TCR affinity (high Koff rate). Two distinct phenotypes were identified at the peak of the response, the first indicating a terminally differentiated subset responsible for IFNγ production and resembling a NKT gene expression profile. A second HCV-CTL was found with low IFNγ production, and dominated by poorly differentiated memory T cells, with a distinct gene signature highly clustered with memory cells identified two years post-infection. This systems approach provides new routes to investigate unresolved T cell dynamics and to identify new mechanisms for exploitation in vaccine development.
(1) Eltahla et al. Immun. Cell Biology 2016 doi:10.1038/icb.2016.16.